Pancreatic islet transcription factor and uses thereof

ABSTRACT

The present invention provides a pancreatic islet transcription factor and methods of treating and diagnosing diabetes.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 10/533,593, filed May 2, 2005, which is a US national phaseapplication of PCT/US2003/036131, filed Nov. 13, 2003, which claimsbenefit of priority to U.S. Provisional Patent Application No.60/425,968, filed Nov. 13, 2002, each of which is incorporated byreference in their entirety for all purposes.

BACKGROUND OF THE INVENTION

Insulin, a hormone required for metabolic homeostasis, is produced onlyin the pancreatic beta cell. Type I Diabetes is characterized by a rapidloss of beta cell mass and a sharp decrease in pancreatic insulincontent. A decline in beta cell mass and function is also characteristicof Type II Diabetes. Intervention to improve beta cell mass and functionis a major goal of diabetes therapeutics.

Perturbations in the expression or function of numerous islettranscription factors have been demonstrated to cause beta celldysfunction. In humans, mutations in islet transcription factorsHNF4alpha, HNF4beta, HNF1 alpha, and pdx-1 lead to Maturity OnsetDiabetes of the Young (MODY) syndromes via the resulting deficit in betacell function. See, e.g., Froguel, P & Velho, G, Trends Endocrinol Metab10: 142-146 (1999)). Functional deletion of other islet transcriptionfactors (e.g. nkx6.1, nkx2.2, is1-1, neuroD/beta2, PAX6) in mice alsolead to beta cell and insulin deficits and diabetes. See, e.g., Melloul,D. et al., Diabetes 45: 309-326 (2002); Sander M et al., Genes Dev 11:1662-1673 (1997); Sander M et al., Development 127: 5533-5540 (2000).Overexpression of pdx1 in islets can lead to restoration of beta cellmass and function in the context of diabetogenic mutations in otherproteins (Kushner J A et al., J Clin Invest 109: 1193-1201 (2002)).Overexpression of pdx-1 and is1-1 in enteric stem cell populationsconfers on them the ability to produce insulin (Kojima H et al.,Diabetes 51: 1398-1408 (2002)). It is clear that the proper complementof islet transcription factors is crucial for maintenance of a stablemetabolic state, and that, in some cases, it is possible to confer oncertain cells the ability to express insulin by providing for increasedexpression of islet transcription factors.

There are five known RFX genes in humans. RFX1, RFX2, RFX3 and RFX4share conserved DNA-binding domains, dimerization domains, and domainsof undefined function called B and C. The dimerization domains allowRFX1-4 proteins to homo- and heterodimerize. This dimerization functionis involved in the transcriptional function of these proteins. RFX 1, 2and 3 also have an additional conserved A domain. RFX5 has the conservedDBD, but lacks the dimerization and A, B, and C domains. RFX5 controlstranscription from the MHC class II gene promoter, and mutations in RFX5lead to the bare lymphocyte syndrome, a serious immunodeficiencydisorder (Reith W and Mach B, Annu. Rev. Immunol. 19: 331-373 (2001)).

The molecular mechanisms that control a cell's ability to produceinsulin remains poorly understood. The present invention addresses thisand other problems.

BRIEF SUMMARY OF THE INVENTION

The present invention provides an isolated nucleic acid encoding anIC-RFX polypeptide at least 70% identical to SEQ ID NO:2. In someembodiments, the nucleic acid encodes SEQ ID NO:2. In some embodiments,the nucleic acid comprises SEQ ID NO:1.

The present invention also provides an isolated nucleic acid encoding apolypeptide comprising in the following order: a proline/glutamine richdomain, an RFX DNA binding domain (SEQ ID NO:4), an RFX B domain (SEQ IDNO:5), an RFX C domain (SEQ ID NO:6), a dimerization domain (SEQ IDNO:7) and a serine/threonine domain.

The present invention also provides an expression cassette comprising apromoter operably linked to the nucleic acid encoding an IC-RFXpolypeptide at least 70% identical to SEQ ID NO:2.

The present invention also provides an isolated nucleic acid thatspecifically hybridizes following at least one wash in 0.2×SSC at 55° C.for 20 minutes to a probe comprising SEQ ID NO:1.

The present invention also provides an isolated IC-RFX polypeptidecomprising an amino acid sequence at least 70% identical to SEQ ID NO:2.In some embodiments, the polypeptide comprises SEQ ID NO:2. In someembodiments, the polypeptide specifically binds to antibodies generatedagainst SEQ ID NO:2.

The present invention also provides an antibody that specifically bindsto SEQ ID NO:2.

The present invention also provides a host cell transfected with thenucleic acid encoding an IC-RFX polypeptide at least 70% identical toSEQ ID NO:2. In some embodiments, the cell is a pancreatic islet cell.In some embodiments, the cell is an islet β-cell.

The present invention also provides methods of diagnosing a subject withdiabetes or a susceptibility for diabetes. In some embodiments, themethods comprise detecting in a sample from the subject a polynucleotidethat hybridizes to a probe comprising SEQ ID NO:1 following at least onewash in 0.2×SSC at 55° C. for 20 minutes. In some embodiments, thepolynucleotide is detected by hybridization. In some embodiments, thepolynucleotide is detected by amplification of the polynucleotide. Insome embodiments, the nucleotide sequence of the polynucleotide isdetermined.

In some embodiments, the method comprises detecting the level of anIC-RFX polypeptide or transcript encoding the IC-RFX polypeptide in asample from the subject, wherein a modulated level of the polypeptide ortranscript in the sample compared to a level of the polypeptide ortranscript in a non-diabetic individual indicates that the subject isdiabetic or is predisposed for at least some pathological aspects ofdiabetes, and wherein the IC-RFX polypeptide is at least 70% identicalto SEQ ID NO:2. In some embodiments, the polypeptide comprises SEQ IDNO:2. In some embodiments, the polypeptide is detected by an antibody.

The invention also provides methods for identifying an agent fortreating a diabetic or pre-diabetic individual. In some embodiments, themethod comprises the steps of (i) contacting an agent to a mixturecomprising an IC-RFX polypeptide at least 70% identical to SEQ ID NO:2;and (ii) selecting an agent that modulates the expression or activity ofthe polypeptide or that binds to the polypeptide. In some embodiments,the method further comprising selecting an agent that modulates insulinexpression of a cell. In some embodiments, step (ii) comprises selectingan agent that modulates expression of the polypeptide. In someembodiments, step (ii) comprises selecting an agent that modulates theactivity of the polypeptide. In some embodiments, step (ii) comprisesselecting an agent that specifically binds to the polypeptide. In someembodiments, the polypeptide is expressed in a cell and the cell iscontacted with the agent. In some embodiments, the polypeptide is SEQ IDNO:2.

The present invention also provides methods of treating a diabetic orpre-diabetic animal. In some embodiments, the methods compriseadministering to the animal a therapeutically effective amount of anagent identified by (i) contacting an agent to a mixture comprising anIC-RFX polypeptide at least 70% identical to SEQ ID NO:2; and (ii)selecting an agent that modulates the expression or activity of thepolypeptide or that binds to the polypeptide. In some embodiments, theagent is an antibody. In some embodiments, the antibody is a monoclonalantibody. In some embodiments, the animal is a human.

The present invention also provides methods of introducing an expressioncassette into a cell. In some embodiments, the methods compriseintroducing into the cell an expression cassette comprising a promoteroperably linked to a polynucleotide encoding an IC-RFX polypeptide atleast 70% identical to SEQ ID NO:2. In some embodiments, the polypeptidecomprises SEQ ID NO:2. In some embodiments, the polynucleotide comprisesSEQ ID NO:1. In some embodiments, the method further comprisingintroducing the cell into a human. In some embodiments, the human isdiabetic. In some embodiments, the human is prediabetic. In someembodiments, the cell is from the human. In some embodiments, the cellis a pancreatic islet cell. In some embodiments, the cell is an islet1-cell.

The present invention also provides for dimers comprising at least oneIC-RFX polypeptide. In some embodiments, the dimer is a homodimer ofIC-RFD polypeptides. In some embodiments, the dimer is a heterodimercomprising one IC-RFX polypeptide and a second polypeptide. In theseembodiments, the second polypeptide is selected from an RFXtranscription factor, e.g., RFX1, RFX2, RFX3, RFX4 or RFX5.

DEFINITIONS

An “IC-RFX transcription factor” or IIC-RFX polypeptide” refers to apolypeptide that is a member of the RFX transcription factor family andhas a structure as illustrated in FIG. 4. IC-RFX polypeptides comprisedomains in the following order: a proline/glutamine rich domain, an RFXDNA binding domain, an RFX B domain, an RFX C domain, a dimerizationdomain and a serine/threonine domain. An exemplary consensus sequencefor IC-RFX polypeptides is SEQ ID NO:3. Exemplary IC-RFX polypeptidesare substantially identical to SEQ ID NO:2.

A “proline/glutamine” domain as used herein refers to an amino acidsubsequence between about 40 to about 150 amino acids, and often between60-90 amino acids, that comprise at least about 8% or at least about 10%proline or glutamine residues, i.e., the sum of proline and glutamineresidues make up at least 8 or 10% of the amino acids in the domain.

An “RFX DNA binding domain” as used herein, refers to an amino acidsubsequence of between about 50 and about 100 amino acids that has asequence substantially identical to SEQ ID NO:4 or conservativesubstitutions thereof. Exemplary RFX DNA binding domains include, e.g.,TLQWLEENYIVCEGVCLPRCILYAHYLDFCRKEKLEPACAATFGKTIRQKFPLLTTRRLGTRGHSKYHYYGIGIKE (SEQ ID NO:8).

An “RFX B domain,” as used herein, refers to an amino acid subsequenceof between about 20 and about 60 amino acids that has a sequencesubstantially identical to SEQ ID NO:5 or conservative substitutionsthereof. Exemplary RFX B domains include, e.g.,KVDTLIMMYKTHCQCILDNAINGNFEEIQHFLLHFW (SEQ ID NO:9).

A “RFX C domain,” as used herein, refers to an amino acid subsequence ofbetween about 20 and about 60 amino acids that has a sequencesubstantially identical to SEQ ID NO:6 or conservative substitutionsthereof. Exemplary RFX C domains include, e.g.,LYKVLTDVLIPATMQEMPESLLADIRNFAKNWEQWVVSSL (SEQ ID NO:10).

A “dimerization domain,” as used herein, refers to an amino acidsubsequence of between about 150 and about 200 amino acids that has asequence substantially identical to SEQ ID NO:7 or conservativesubstitutions thereof. Exemplary RFX dimerization domains include, e.g.,RFVSSLKRQTSFLHLAQIARPALFDQHVVNSMVSDIERVDLNSIGSQALLTISGSTDTESGIYTEHDSITVFQELKDLLKKNATVEAFIEWLDTVVEQRVIKTSKQNGRSLKKRAQDFLLKWSFFGARVMHNLTLNNASSFGSFHLIRMLLDEYILLAMETQFNNDKEQELQN LLDKYM (SEQ IDNO:11).

A “serine/threonine” domain as used herein refers to an amino acidsubsequence between about 50 to about 200 amino acids, and often between100-500 amino acids, that comprises at least about 15% and sometimes atleast about 20% proline or glutamine residues.

An “IC-RFX nucleic acid” or an “IC-RFX polynucleotide” refers to anucleic acid or polynucleotide encoding an IC-RFX polypeptide orfragment thereof. Exemplary IC-RFX polynucleotides include, e.g., SEQ IDNO:1.

A “beta cell phenotype” refers to the expression of markers thatnormally distinguish the beta cells from the other pancreatic isletscells. For example, beta cells express insulin in a glucose dependentmanner, express Nkx6.1 or glucokinase.

A “lean individual,” when used to compare with a sample from a patient,refers to an adult with a fasting blood glucose level less than 110mg/dl or a 2 hour PG reading of 140 mg/dl. “Fasting” refers to nocaloric intake for at least 8 hours. A “2 hour PG” refers to the levelof blood glucose after challenging a patient to a glucose loadcontaining the equivalent of 75 g anhydrous glucose dissolved in water.The overall test is generally referred to as an oral glucose tolerancetest (OGTT). See, e.g., Diabetes Care, Supplement 2002, AmericanDiabetes Association: Clinical Practice Recommendations 2002. The levelof a polypeptide in a lean individual can be a reading from a singleindividual, but is typically a statistically relevant average from agroup of lean individuals. The level of a polypeptide in a leanindividual can be represented by a value, for example in a computerprogram.

A “pre-diabetic individual,” when used to compare with a sample from apatient, refers to an adult with a fasting blood glucose level greaterthan 110 mg/dl but less than 126 mg/dl or a 2 hour PG reading of greaterthan 140 mg/dl but less than 200 mg/dl. A “diabetic individual,” whenused to compare with a sample from a patient, refers to an adult with afasting blood glucose level greater than 126 mg/dl or a 2 hour PGreading of greater than 200 mg/dl.

“Antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof whichspecifically bind and recognize an analyte (antigen). The recognizedimmunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially an Fab withpart of the hinge region (see, Paul (Ed.) Fundamental Immunology, ThirdEdition, Raven Press, NY (1993)). While various antibody fragments aredefined in terms of the digestion of an intact antibody, one of skillwill appreciate that such fragments may be synthesized de novo eitherchemically or by utilizing recombinant DNA methodology. Thus, the termantibody, as used herein, also includes antibody fragments eitherproduced by the modification of whole antibodies or those synthesized denovo using recombinant DNA methodologies (e.g., single chain Fv).

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

The term “isolated,” when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is essentially free of other cellularcomponents with which it is associated in the natural state. It ispreferably in a homogeneous state although it can be in either a dry oraqueous solution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinthat is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames that flank the gene and encode a protein otherthan the gene of interest. The term “purified” denotes that a nucleicacid or protein gives rise to essentially one band in an electrophoreticgel. Particularly, it means that the nucleic acid or protein is at least85% pure, more preferably at least 95% pure, and most preferably atleast 99% pure.

The term “nucleic acid” or “polynucleotide” refers todeoxyribonucleotides or ribonucleotides and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions) andcomplementary sequences as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka etal., J. Biol. Chem. 260:2605-2608 (1985); and Cassol et al. (1992);Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleicacid is used interchangeably with gene, cDNA, and mRNA encoded by agene.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers. As usedherein, the terms encompass amino acid chains of any length, includingfull-length proteins (i.e., antigens), wherein the amino acid residuesare linked by covalent peptide bonds.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. “Amino acid mimetics” refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but which functions in amanner similar to a naturally occurring amino acid.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein that encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins (1984)).

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95%identity over a specified region), when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. Such sequences are then said tobe “substantially identical.” This definition also refers to thecomplement of a test sequence. Optionally, the identity exists over aregion that is at least about 50 nucleotides in length, or morepreferably over a region that is 100 to 500 or 1000 or more nucleotidesin length.

The term “similarity,” or “percent similarity,” in the context of two ormore polypeptide sequences, refer to two or more sequences orsubsequences that have a specified percentage of amino acid residuesthat are either the same or similar as defined in the 8 conservativeamino acid substitutions defined above (i.e., 60%, optionally 65%, 70%,75%, 80%, 85%, 90%, or 95% similar over a specified region), whencompared and aligned for maximum correspondence over a comparisonwindow, or designated region as measured using one of the followingsequence comparison algorithms or by manual alignment and visualinspection. Such sequences are then said to be “substantially similar.”Optionally, this identity exists over a region that is at least about 50amino acids in length, or more preferably over a region that is at leastabout 100 to 500 or 1000 or more amino acids in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443,by the search for similarity method of Pearson and Lipman (1988) Proc.Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., Ausubelet al., Current Protocols in Molecular Biology (1995 supplement)).

Examples of an algorithm that is suitable for determining percentsequence identity and sequence similarity include the BLAST and BLAST2.0 algorithms, which are described in Altschul et al. (1977) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g., greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. For selective or specific hybridization, apositive signal is at least two times background, optionally 10 timesbackground hybridization. Exemplary stringent hybridization conditionscan be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42°C., or 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and0.1% SDS at 65° C. Such washes can be performed for 5, 15, 30, 60, 120,or more minutes.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides thatthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. Such washes can be performed for 5, 15,30, 60, 120, or more minutes. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency.

The phrase “a nucleic acid sequence encoding” refers to a nucleic acidthat contains sequence information for a structural RNA such as rRNA, atRNA, or the primary amino acid sequence of a specific protein orpeptide, or a binding site for a trans-acting regulatory agent. Thisphrase specifically encompasses degenerate codons (i.e., differentcodons which encode a single amino acid) of the native sequence orsequences that may be introduced to conform with codon preference in aspecific host cell.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (nonrecombinant) form of the cell or expressnative genes that otherwise are expressed abnormally, under-expressed ornot expressed at all.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

The phrase “specifically (or selectively) binds to an antibody” or“specifically (or selectively) immunoreactive with”, when referring to aprotein or peptide, refers to a binding reaction which is determinativeof the presence of the protein in the presence of a heterogeneouspopulation of proteins and other biologics. Thus, under designatedimmunoassay conditions, the specified antibodies bind to a particularprotein and do not bind in a significant amount to other proteinspresent in the sample. Specific binding to an antibody under suchconditions may require an antibody that is selected for its specificityfor a particular protein. For example, antibodies raised against aprotein having an amino acid sequence encoded by any of thepolynucleotides of the invention can be selected to obtain antibodiesspecifically immunoreactive with that protein and not with otherproteins, except for polymorphic variants. A variety of immunoassayformats may be used to select antibodies specifically immunoreactivewith a particular protein. For example, solid-phase ELISA immunoassays,Western blots, or immunohistochemistry are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. See,Harlow and Lane Antibodies, A Laboratory Manual, Cold Spring HarborPublications, NY (1988) for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity.Typically, a specific or selective reaction will be at least twice thebackground signal or noise and more typically more than 10 to 100 timesbackground.

“Inhibitors,” “activators,” and “modulators” of IC-RFX expression or ofIC-RFX activity are used to refer to inhibitory, activating, ormodulating molecules, respectively, identified using in vitro and invivo assays for IC-RFX expression or IC-RFX activity, e.g., ligands,agonists, antagonists, and their homologs and mimetics. The term“modulator” includes inhibitors and activators. Inhibitors are agentsthat, e.g., inhibit expression of IC-RFX or bind to, partially ortotally block stimulation or enzymatic activity, decrease, prevent,delay activation, inactivate, desensitize, or down regulate the activityof IC-RFX, e.g., antagonists. Activators are agents that, e.g., induceor activate the expression of IC-RFX or bind to, stimulate, increase,open, activate, facilitate, enhance activation or enzymatic activity,sensitize or up regulate the activity of IC-RFX, e.g., agonists.Modulators include naturally occurring and synthetic ligands,antagonists, agonists, small chemical molecules and the like. Suchassays for inhibitors and activators include, e.g., applying putativemodulator compounds to pancreatic cells or other cells expressingIC-RFX, in the presence or absence of IC-RFX modulators and thendetermining the functional effects on IC-RFX activity, as describedabove. Samples or assays comprising IC-RFX that are treated with apotential activator, inhibitor, or modulator are compared to controlsamples without the inhibitor, activator, or modulator to examine theextent of effect. Control samples (untreated with modulators) areassigned a relative IC-RFX activity value of 100%. Inhibition of IC-RFXis achieved when the IC-RFX activity value relative to the control isabout 80%, optionally 50% or 25-1%. Activation of IC-RFX is achievedwhen the IC-RFX activity value relative to the control is 110%,optionally 150%, optionally 200-500%, or 1000-3000% higher.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates that human IC-RFX is highly enriched in pancreaticislets as demonstrated by GeneChip data. Average Difference values froma survey of identical quantities of human tissue RNAs were obtained byhybridization of samples to a custom human islet Affymetrix GeneChiparray. Probe set RD_BG02340, which detects IC-RFX transcripts, displaysstrong hybridization signals with human islet RNA samples from 5separate donor pancreases (HP509, Hislet, HP384-7, HP741-1 and Htype2)but no hybridization signal to any of ten other human tissues (pancreas,adipose, brain, heart, kidney, liver, lung, skeletal muscle, smallintestine or thymus).

FIG. 2 illustrates that human IC-RFX is highly enriched in pancreaticislets as demonstrated by Northern blot. A ³²P-labeled human IC-RFX cDNAprobe hybridized with filters A and B simultaneously under identicalconditions. Filter A is a blot of 10 μg of total RNA from a human isletsample along with 2 μg polyA+ RNAs from two different human pancreassamples, as well as 2 μg samples of polyA+ RNA from human testis,adrenal and spleen. Filter B is a commercial Multiple Tissue Northernblot (Clontech 2001 catalog #7780-1). A hybridizing transcript(approximately 3.3 kb) is found only in the human islet sample.

FIG. 3 illustrates that mouse IC-RFX is highly enriched in pancreaticislets. Average Difference values from a survey of identical quantitiesof mouse tissue RNAs or mouse beta cell lines were obtained byhybridization of samples to a custom mouse islet Affymetrix GeneChiparray. Probe set MBXMUSISL22609, which detects IC-RFX transcripts,displays strong hybridization signals with two samples of a mouse betacell line (betaHC9_(—)1, betaHC9_(—)2) and four separate mouse islet RNAsamples multiple different mice (islet7, islet10, islet11, islet12) butno hybridization signal to any of ten other mouse tissues (adipose,brain, heart, kidney, liver, lung, pituitary, skeletal muscle, smallintestine, thymus).

FIG. 4 provides a domain structure comparison of IC-RFX with other RFXproteins. DBD=DNA binding domain, DIM=dimerization domain, A, B and Cdesignate conserved domains of unknown function,PQ=proline/glutamine-rich region, ST=serine/threonine-rich region,Q=glutamine-rich region, P=proline-rich region, andDE=glutamate/aspartate-rich region. Sak1 is the S. pombe RFXrepresentative, Daf-19 is a C. elegans gene product, and DmRFX is aDrosophila RFX protein.

FIG. 5 illustrates an amino acid sequence alignment of the DNA bindingdomains of IC-RFX (SEQ ID NO:8) and other mammalian RFX proteins (SEQ IDNOS:12, 13 and 15-17) and the C. elegans RFX protein daf-19 (SEQ IDNO:14). A consensus sequence is shown at the bottom of each alignment(SEQ ID NOS:18-21). Asterisks mark the residues involved in DNA bindingin RFX1. Boxed and shaded residues define broadly conserved residues.

FIG. 6 illustrates an amino acid sequence alignment of the B domains ofIC-RFX (SEQ ID NO:9) and other mammalian RFX proteins (SEQ ID NOS:23-26)and the C. elegans RFX protein daf-19 (SEQ ID NO:22). A consensussequence is shown at the bottom of each alignment. Boxed and shadedresidues define broadly conserved residues.

FIG. 7 illustrates an amino acid sequence alignment of the C domains ofIC-RFX (SEQ ID NO:10) and other mammalian RFX proteins (SEQ ID NOS:27and 28) and the C. elegans RFX protein daf-19 (SEQ ID NO:29). Aconsensus sequence is shown at the bottom of each alignment. Boxed andshaded residues define broadly conserved residues.

FIG. 8 illustrates an amino acid sequence alignment of the dimerizationdomains of IC-RFX (SEQ ID NO:11) and other mammalian RFX proteins (SEQID NOS:30, 32 and 33) and the C. elegans.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present application provides a novel islet-specific RFXtranscription factor, designated IC-RFX, as well as methods andcompositions useful for diagnosis and treatment of diabetes. Thus, theinvention provides for diagnostic assays for detecting mutations in theIC-RFX gene, thereby diagnosing diabetics or those pre-disposed todevelop diabetes. In addition, the invention provides for monitoring ofexpression of IC-RFX RNA or polypeptides.

Moreover, the invention provides for methods of supplementing IC-RFXexpression in islets or other cells to promote differentiation of thecells into function beta-cells (i.e., insulin producing cells). Theinvention further provides for ex vivo supplementation or expression ofIC-RFX in cells to promote beta cell differentiation prior totransplantation into a host.

IC-RFX also provides a useful target to screen for drugs for treatingdiabetes and increasing insulin production.

II. IC-RFX Expression in Cells and Induction of Insulin Production andBeta-Cell Differentiation

Pancreatic beta-cells can be produced from non-beta cell pancreaticcells by providing for production of IC-RFX in a pancreatic cell eitherin vivo (e.g., by administration of an IC-RFX-encoding nucleic acid(e.g., RNA or DNA) to the pancreas of a subject, e.g., by introductionof nucleic acid into a lumen of a pancreatic duct), or in vitro, e.g.,by contacting a target cell (e.g., an isolated, non-beta, pancreaticcell) with an IC-RFX-encoding nucleic acid (e.g., RNA or DNA) in culture(which cells can then be cultured, expanded, and transplanted into asubject). In some embodiments, other islet specific transcriptionfactors such as neurogenin1, neurogenin2, neurogenin3, NeuroD1/BETA2,PDX-1, HNF4alpha, HNF4beta, HNF1alpha, nkx6.1, nkx2.2, is1-1, PAX6,Pax4, neuroD4, pbx-1, HB9, HNF6, HNF3b/FoxA2, or dimerization partnersof IC-RFX, such as RFX1, RFX2, RFX3, RFX4 or RFX5, are also expressed inthe same cell to induce differentiation and insulin production.

In one embodiment, beta cells are produced by providing for expressionof IC-RFX at a level sufficient to induce a beta cell phenotype (e.g.,glucose-dependent expression of insulin, glucokinase expression, etc.)in the target cell. Expression of IC-RFX in the target cell can beaccomplished in a variety of ways. For example, in one embodiment,IC-RFX expression is accomplished by introduction of an IC-RFX-encodingnucleic acid (e.g., DNA or RNA) to provide for expression of the encodedIC-RFX polypeptide in the target cell). In another embodiment, IC-RFXexpression is induced by introduction of a gene encoding a protein thatprovides for induction of IC-RFX expression (e.g., expression of an“upstream” positive regulator of IC-RFX expression in the target cell).In another embodiment, IC-RFX expression is accomplished by introductionof a gene encoding a protein that inhibits activity (e.g., function orexpression) a negative regulator of IC-RFX expression. In anotherembodiment, IC-RFX expression is induced by introduction of a smallmolecule that provides for induction of IC-RFX expression (e.g., a smallmolecule pharmaceutical that induces IC-RFX expression in the targetcell). In addition, production of pancreatic beta cells of the inventioncan also be accomplished by providing for production of factors inducedby IC-RFX.

Where the IC-RFX nucleic acid to be delivered is DNA, any constructhaving a promoter (e.g., a promoter that is functional in a eukaryoticcell) operably linked to a DNA of interest can be used in the invention.Constructs for use in the invention may be any eukaryotic expressionconstruct containing the IC-RFX DNA or RNA sequence of interest.Typically, the construct is capable of replication in eukaryotic and/orprokaryotic hosts (viruses in eukaryotic, plasmids in prokaryotic),which constructs are known in the art and are commercially available.

The constructs can be prepared using techniques well known in the art.Likewise, techniques for obtaining expression of exogenous DNA or RNAsequences in a genetically altered host cell are known in the art (see,for example, Kormal et al., Proc. Natl. Acad. Sci. USA, 84:2150-2154(1987); Sambrook et al. M OLECULAR CLONING: A LABORATORY MANUAL, 2ndEd., (1989).

In one embodiment, the DNA construct contains a promoter to facilitateexpression of the DNA of interest within a pancreatic cell. The promotercan be a strong, viral promoter that functions in eukaryotic cells,e.g., a promoter from cytomegalovirus (CMV), mouse mammary tumor virus(MMTV), Rous sarcoma virus (RSV), or adenovirus. More specifically,exemplary promoters include the promoter from the immediate early geneof human CMV (Boshart et al., Cell 41:521-530 919850) and the promoterfrom the long terminal repeat (LTR) of RSV (Gorman et al., Proc. Natl.Acad. Sci. USA 79:6777-6781 (1982)).

Alternatively, the promoter used may be a strong general eukaryoticpromoter such as the actin gene promoter. In one embodiment, thepromoter used may be a tissue-specific promoter. For example, thepromoter used in the construct may be a pancreas specific promoter, aduct cell specific promoter or a stem cell specific promoter. Inaddition to promoters, the constructs of the invention can includesequences that enhance expression in the target cells. In anotherembodiment, the promoter is a regulated promoter, such as atetracycline-regulated promoter, expression from which can be regulatedby exposure to an exogenous substance (e.g., tetracycline).

The constructs can also comprise other components such as a marker(e.g., an antibiotic resistance gene (such as an ampicillin resistancegene) or β-galactosidase) to aid in selection or identification of cellscontaining and/or expressing the construct, an origin of replication forstable replication of the construct in a bacterial cell (preferably, ahigh copy number origin of replication), a nuclear localization signal,or other elements which facilitate production of the DNA construct, theprotein encoded, or both.

For eukaryotic expression, the construct can contain a eukaryoticpromoter operably linked to a DNA of interest, which is in turn operablylinked to a polyadenylation signal sequence. The polyadenylation signalsequence may be selected from any of a variety of polyadenylation signalsequences known in the art. An exemplary polyadenylation signal sequenceis the SV40 early polyadenylation signal sequence. The construct mayalso include one or more introns, where appropriate, which can increaselevels of expression of the DNA of interest, particularly where the DNAof interest is a cDNA (e.g., contains no introns of thenaturally-occurring sequence). Any of a variety of introns known in theart may be used.

In an alternative embodiment, the nucleic acid delivered to the cell isan RNA encoding an IC-RFX polypeptide. In this embodiment, the RNA isadapted for expression (i.e., translation of the RNA) in a target cell.Methods for production of RNA (e.g., mRNA) encoding a protein ofinterest are well known in the art, and can be readily applied toproduce RNA encoding IC-RFX polypeptides of the invention.

Delivery of IC-RFX nucleic acids can be accomplished using a viral or anon-viral vector. In one embodiment the nucleic acid is delivered withina viral particle, such as an adenovirus. In another embodiment, thenucleic acid is delivered in a formulation comprising naked DNA admixedwith an adjuvant such as viral particles (e.g., adenovirus) or cationiclipids or liposomes. The precise vector and vector formulation used willdepend upon several factors, such as the size of the DNA to betransferred, the delivery protocol to be used, and the like.

In general, viral vectors used in accordance with the invention arecomposed of a viral particle derived from a naturally-occurring viruswhich has been genetically altered to render the virusreplication-defective and to deliver a recombinant gene of interest forexpression in a target cell in accordance with the invention.

Numerous viral vectors are well known in the art, including, forexample, retrovirus, adenovirus, adeno-associated virus, herpes simplexvirus (HSV), cytomegalovirus (CMV), vaccinia and poliovirus vectors.Adenovirus and AAV are usually preferred viral vectors since theseviruses efficiently infect slowly replicating and/or terminallydifferentiated cells. The viral vector may be selected according to itspreferential infection of the cells targeted.

Where a replication-deficient virus is used as the viral vector, theproduction of infectious virus particles containing either DNA or RNAcorresponding to the DNA of interest can be achieved by introducing theviral construct into a recombinant cell line that provides the missingcomponents essential for viral replication. In one embodiment,transformation of the recombinant cell line with the recombinant viralvector will not result in production or substantial production ofreplication-competent viruses. Methods for production ofreplication-deficient viral particles containing a nucleic acid ofinterest are well known in the art and are described in, for example,Rosenfeld et al., Science 252:431-434 (1991) and Rosenfeld et al., Cell68:143-155 (1992; U.S. Pat. No. 5,139,941; U.S. Pat. No. 4,861,719; andU.S. Pat. No. 5,356,806. Methods and materials for manipulation of themumps virus genome, characterization of mumps virus genes responsiblefor viral fusion and viral replication, and the structure and sequenceof the mumps viral genome are described in Tanabayashi et al., J. Virol.67:2928-2931 (1993); Takeuchi et al., Archiv. Virol., 128:177-183(1993); Tanabayashi et al., Virol. 187:801-804 (1992); Kawano et al.,Virol., 179:857-861 (1990); Elango et al., J. Gen. Virol. 69:2893-28900(1988).

The nucleic acid of interest can also be introduced into a cell using anon-viral vector. Non-viral vectors include naked DNA (e.g., DNA notcontained within a viral particle, and free of a carrier molecules suchas lipids), chemical formulations comprising naked nucleic acid (e.g., aformulation of DNA (and/or RNA) and cationic compounds (e.g., dextransulfate, cationic lipids)), and naked nucleic acid mixed with anadjuvant such as a viral particle (e.g., the DNA of interest is notcontained within the viral particle, but the formulation is composed ofboth naked DNA and viral particles (e.g., adenovirus particles).

Alternatively or in addition, the nucleic acid can be complexed withpolycationic substances such as poly-L-lysine or DEAC-dextran, targetingligands, and/or DNA binding proteins (e.g., histones). DNA- orRNA-liposome complex formulations comprise a mixture of lipids whichbind to genetic material (DNA or RNA) and facilitate delivery of thenucleic acid into the cell. Liposomes which can be used in accordancewith the invention include DOPE (dioleyl phosphatidyl ethanol amine),CUDMEDA (N-(5-cholestrum-3-β-ol 3-urethanyl)-N′,N′-dimethylethylenediamine).

The nucleic acid of interest can also be administered as a chemicalformulation of DNA or RNA coupled to a carrier molecule (e.g., anantibody or a receptor ligand), which facilitates delivery to host cellsfor the purpose of altering the biological properties of the host cells.Chemical formulations include modifications of nucleic acids that allowcoupling of the nucleic acid compounds to a carrier molecule such as aprotein or lipid, or derivative thereof. Exemplary protein carriermolecules include antibodies specific to the cells of a targetedpancreatic cell or receptor ligands, e.g., molecules capable ofinteracting with receptors associated with a cell of a targetedpancreatic cell.

Nucleic acid encoding an IC-RFX polypeptide can be introduced into acell in vitro to provide for at least transient expression. The cellsinto which the nucleic acid is introduced can be differentiatedepithelial cells (e.g., pancreatic cells, gut cells, hepatic cells orduct cells), pluripotent adult or embryonic stem cells, or any mammaliancell capable of developing into β-cells or cells capable of expressionof insulin in vitro following expression of an IC-RFX-encoding nucleicacid. The cell is subsequently implanted into a subject having adisorder characterized by a deficiency in insulin (e.g., diabetes),which disorder is amenable to treatment by islet cell replacementtherapy. In some embodiments, the transfected host cell implanted in thesubject is derived from the individual who will receive the transplant(e.g., to provide an autologous transplant). For example, in a subjecthaving Type 1 diabetes, pluripotent stem cells, hepatic cells, gut cellsor pancreatic cells can be isolated from the affected subject, the cellsmodified to express IC-RFX-encoding DNA, and the cells implanted in theaffected subject to provide for insulin production, or the transformedcells cultured so as to facilitate development of the cells intoinsulin-producing i-cells. Alternatively, pluripotent stem cells,hepatic cells, gut cells or pancreatic cells from another subject (the“donor”) could be modified to express IC-RFX-encoding DNA, and the cellssubsequently implanted in the affected subject to provide for insulinproduction, or the transformed cells cultured so as to facilitatedevelopment of the cells into insulin-producing .beta.-cells.

Introduction of nucleic acid into the cell in vitro can be accomplishedaccording to methods well known in the art (e.g., through use ofelectroporation, microinjection, lipofection infection with arecombinant (preferably replication-deficient) virus, and other meanswell known in the art). The nucleic acid is generally operably linked toa promoter that facilitates a desired level of polypeptide expression(e.g., a promoter derived from CMV, SV40, adenovirus, or atissue-specific or cell type-specific promoter). Transformed cellscontaining the recombinant nucleic acid can be selected and/or enrichedvia, for example, expression of a selectable marker gene present in theintroduced construct or that is present on a nucleic acid that isco-transfected with the construct. Typically selectable markers providefor resistance to antibiotics such as tetracycline, hygromycin,neomycin, and the like. Other markers can include thymidine kinase andthe like. Other markers can include markers that can be used to identifyexpressing cells, such as beta-galactosidase or green florescentprotein.

Expression of the introduced nucleic acid in the transformed cell can beassessed by various methods known in the art. For example, expression ofthe introduced gene can be examined by Northern blot to detect mRNA thathybridizes with a DNA probe derived from the relevant gene. Those cellsthat express the desired gene can be further isolated and expanded in invitro culture using methods well known in the art. The host cellsselected for transformation will vary with the purpose of the ex vivotherapy (e.g., insulin production), the site of implantation of thecells, and other factors that will vary with a variety of factors thatwill be appreciated by the ordinarily skilled artisan.

The transformed cell can also be examined for the development of anislet cell phenotype. For example, expression of insulin could bedetected by PCR, northern blot, immunocytochemistry, western blot, orELISA. Alternatively a marker gene such as green florescent protein oran antibiotic resistance gene operatively linked to an islet specificpromoter such as the insulin gene promoter could be used foridentification or selection of differentiated islet cells. Methods forengineering a host cell for expression of a desired gene product(s) andimplantation or transplantation of the engineered cells (e.g., ex vivotherapy) are known in the art. See, e.g., Gilbert et al. Transplantation56:423-427 (1993). For expression of a desired gene in exogenous orautologous cells and implantation of the cells (e.g., islet cells) intopancreas, see, e.g., Docherty, Clin Sci (Colch) 92:321-330 (1997); Hegreet al., Acta Endocrinol Suppl (Copenh) 205:257-281 (1976); Sandler etal., Transplantation 63:1712-1718 (1997); Calafiore, Diabetes Care20:889-896 (1997); Kenyon et al., Diabetes Metab Rev 12:361-372 (1996);Chick et al. Science 197:780-782 (1977). In general, the cells can beimplanted into the pancreas, or to any practical or convenient site,e.g., subcutaneous site, liver, peritoneum.

In general, after expansion of the transformed cells in vitro, the cellsare implanted into the mammalian subject by methods well known in theart. The number of cells implanted is a number of cells sufficient toprovide for expression of levels of insulin sufficient to lower bloodglucose levels. The number of cells to be transplanted can be determinedbased upon such factors as the levels of polypeptide expression achievedin vitro, and/or the number of cells that survive implantation. Thetransformed cells are implanted in an area of dense vascularization suchas the liver, and in a manner that minimizes surgical intervention inthe subject. The engraftment of the implant of transformed cells ismonitored by examining the mammalian subject for classic signs of graftrejection, i.e., inflammation and/or exfoliation at the site ofimplantation, and fever, and by monitoring blood glucose levels.

The transplantation method described above is not limited to theexpression of IC-RFX. Engineering a host cell for expression ofadditional transcription factors, such as islet-specific transcriptionfactors (e.g., neurogenin1, neurogenin2, neurogenin3, NeuroD1/BETA2,PDX-1, HNF4alpha, HNF4beta, HNF1alpha, nkx6.1, nkx2.2, is1-1, PAX6,Pax4, neuroD4, pbx-1, HB9, HNF6, HNF3b/FoxA2), or dimerization partnersof IC-RFX (such as RFX1, RFX2, RFX3, RFX4 or RFX5) may be beneficial tosubjects with insulin deficiencies.

IC-RFX-encoding nucleic acids can be delivered directly to a subject toprovide for IC-RFX expression in a target cell (e.g., a pancreatic cell,gut cell, liver cell, or other organ cell capable of expressing anIC-RFX transcription factor and providing production of insulin),thereby promoting development of the cell into an insulin-producing cell(e.g., in pancreas) or to cure a defect in transcription factorexpression in the subject. Methods for in vivo delivery of a nucleicacid of interest for expression in a target cell are known in the art.For example, in vivo methods of gene delivery normally employ either abiological means of introducing the DNA into the target cells (e.g., avirus containing the DNA of interest) or a mechanical means to introducethe DNA into the target cells (e.g., direct injection of DNA into thecells, liposome fusion, or pneumatic injection using a gene gun).

In general terms, the delivery method comprises introducing a nucleicacid into a pancreatic cell. For example, the nucleic acid of interestcan be provided in either a viral or non-viral vector (including nakedDNA) that is introduced into the pancreas in vivo via the duct system.Intraductal administration can be accomplished by cannulation by, forexample, insertion of the cannula through a lumen of thegastrointestinal tract, by insertion of the cannula through an externalorifice, or insertion of the cannula through the common bile duct.Retrograde ductal administration may be accomplished in the pancreas byendoscopic retrograde chalangio-pancreatography (ECRP). Exemplarymethods for accomplishing intraductal delivery to the pancreas aredescribed in U.S. Pat. No. 6,004,944.

The precise amount of IC-RFX-encoding nucleic acid administered willvary greatly according to a number of factors including thesusceptibility of the target cells to transformation, the size andweight of the subject, the levels of protein expression desired, and thecondition to be treated. The amount of nucleic acid and/or the number ofinfectious viral particles effective to infect the targeted tissue,transform a sufficient number of cells, and provide for production of adesired level of insulin can be readily determined based upon suchfactors as the efficiency of the transformation in vitro and thesusceptibility of the targeted cells to transformation. Generally, theamounts of DNA can be extrapolated from the amounts of DNA effective fordelivery and expression of the desired gene in an animal model. Forexample, the amount of DNA for delivery in a human is roughly 100 timesthe amount of DNA effective in a rat.

Pancreatic cells modified according to the invention can facilitatesufficiently high levels of expression of a nucleic acid of interest,particularly where the nucleic acid delivered is DNA and the DNA ofinterest is operably linked to a strong eukaryotic promoter (e.g., CMV,MMTV). The expressed protein can induce islet cell and insulinproduction. Thus the methods of the invention are useful in treating amammalian subject having a variety of insulin related conditions.

The actual number of transformed pancreatic cells required to achievetherapeutic levels of the protein of interest will vary according toseveral factors including the protein to be expressed, the level ofexpression of the protein by the transformed cells, the rate in whichthe protein induces islet cell production (in particular β-cells), andthe condition to be treated.

Regardless of whether the IC-RFX-encoding nucleic acid is introduced invivo or ex vivo, the nucleic acid (or islet cells produced in vitro orrecombinant cells expressing the IC-RFX nucleic acid that are to betransplanted for development into islet cells in vivopost-transplantation) can be administered in combination with othergenes and other agents

The effects of ex vivo or in vivo therapy according to the methods ofthe invention can be monitored in a variety of ways. Generally, a sampleof blood from the subject can be assayed for, for example, levels ofglucose, proinsulin, c-peptide, and insulin. Appropriate assays fordetecting proinsulin, c-peptide, insulin and glucose in blood samplesare well known in the art. Evidence for recurrent autoimmunity can begauged by assaying for autoreactive T cells or for antibodies againstislet proteins such as glutamic acid decarboxylase (GAD), or otherautoantigens well known in the art.

III. General Recombinant Nucleic Acid Methods for Use with the Invention

In numerous embodiments of the present invention, nucleic acids encodingan IC-RFX polypeptide of the present invention will be isolated andcloned using recombinant methods. Such embodiments are used, e.g., toisolate polynucleotides identical or substantially identical to SEQ IDNO:1 for protein expression or during the generation of variants,derivatives, expression cassettes, or other sequences derived from anpolypeptide or polynucleotide of the invention, to monitor geneexpression, for the isolation or detection of sequences in differentspecies, for diagnostic purposes in a patient, e.g., to detect mutationsin a polypeptide or polynucleotide of the invention or to detectexpression levels of nucleic acids or polypeptides. In some embodiments,the sequences encoding the polypeptides of the invention are operablylinked to a heterologous promoter. In one embodiment, the nucleic acidsof the invention are from any mammal, including, in particular, e.g., ahuman, a mouse, a rat, etc.

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

The sequence of the cloned genes and synthetic oligonucleotides can beverified after cloning using, e.g., the chain termination method forsequencing double-stranded templates of Wallace et al., Gene 16:21-26(1981).

In general, the nucleic acids encoding IC-RFX polypeptides are clonedfrom DNA sequence libraries that are made to encode cDNA or genomic DNA.The particular sequences can be located by hybridizing with anoligonucleotide probe, the sequence of which can be derived from thesequences disclosed herein, which provide a reference for PCR primersand defines suitable regions for isolating probes specific for thepolypeptides or polynucleotides of the invention. Alternatively, wherethe sequence is cloned into an expression library, the expressedrecombinant protein can be detected immunologically with antisera orpurified antibodies made against a polypeptide of interest, includingthose disclosed herein.

Methods for making and screening genomic and cDNA libraries are wellknown to those of skill in the art (see, e.g., Gubler and Hoffman Gene25:263-269 (1983); Benton and Davis Science, 196:180-182 (1977); andSambrook, supra).

Briefly, to make the cDNA library, one should choose a source that isrich in the desired cDNA, e.g., pancreatic or islet cells. The mRNA canthen be made into cDNA, ligated into a recombinant vector, andtransfected into a recombinant host for propagation, screening andcloning. For a genomic library, the DNA is extracted from a suitabletissue and either mechanically sheared or enzymatically digested toyield fragments of preferably about 5-100 kb. The fragments are thenseparated by gradient centrifugation from undesired sizes and areconstructed in bacteriophage lambda vectors. These vectors and phage arepackaged in vitro, and the recombinant phages are analyzed by plaquehybridization. Colony hybridization is carried out as generallydescribed in Grunstein et al., Proc. Natl. Acad. Sci. USA., 72:3961-3965(1975).

An alternative method combines the use of synthetic oligonucleotideprimers with polymerase extension on an mRNA or DNA template. Suitableprimers can be designed from specific sequences disclosed herein. Thispolymerase chain reaction (PCR) method amplifies the nucleic acidsencoding the protein of interest directly from mRNA, cDNA, genomiclibraries or cDNA libraries. Restriction endonuclease sites can beincorporated into the primers. Polymerase chain reaction or other invitro amplification methods may also be useful, for example, to clonenucleic acids encoding specific proteins and express said proteins, tosynthesize nucleic acids that will be used as probes for detecting thepresence of mRNA encoding a polypeptide of the invention inphysiological samples, for nucleic acid sequencing, or for otherpurposes (see, U.S. Pat. Nos. 4,683,195 and 4,683,202). Genes amplifiedby a PCR reaction can be purified from agarose gels and cloned into anappropriate vector.

Appropriate primers and probes for identifying the genes encoding apolypeptide of the invention from mammalian tissues can be derived fromthe sequences provided herein. For a general overview of PCR, see, Inniset al. PCR Protocols: A Guide to Methods and Applications, AcademicPress, San Diego (1990).

Synthetic oligonucleotides can be used to construct genes. This is doneusing a series of overlapping oligonucleotides, usually 40-120 bp inlength, representing both the sense and anti-sense strands of the gene.These DNA fragments are then annealed, ligated and cloned.

A polynucleotide encoding a polypeptide of the invention can be clonedusing intermediate vectors before transformation into mammalian cellsfor expression. These intermediate vectors are typically prokaryotevectors or shuttle vectors. The proteins can be expressed in eitherprokaryotes or eukaryotes, using standard methods well known to those ofskill in the art.

Expression cassettes comprising a promoter operably linked to apolynucleotide encoding an IC-RFX polypeptide can be constructed withstandard molecular methods. In some cases, the promoter is heterologousto the polynucleotide.

IV. Purification of Proteins of the Invention

Either naturally occurring or recombinant polypeptides of the inventioncan be purified for use in functional assays. Naturally occurringpolypeptides of the invention can be purified from any source (e.g.,from islet cells or other tissues expressing IC-RFX). Recombinantpolypeptides can be purified from any suitable expression system.

The polypeptides of the invention may be purified to substantial purityby standard techniques, including selective precipitation with suchsubstances as ammonium sulfate; column chromatography,immunopurification methods, and others (see, e.g., Scopes, ProteinPurification Principles and Practice (1982); U.S. Pat. No. 4,673,641;Ausubel et al., supra; and Sambrook et al., supra).

A number of procedures can be employed when recombinant polypeptides arebeing purified. For example, proteins having established molecularadhesion properties can be reversibly fused to a polypeptide of theinvention. With the appropriate ligand, either protein can beselectively adsorbed to a purification column and then freed from thecolumn in a relatively pure form. The fused protein may be then removedby enzymatic activity. Finally polypeptides can be purified usingimmunoaffinity columns.

A. Purification of Proteins from Recombinant Bacteria

When recombinant proteins are expressed by the transformed bacteria inlarge amounts, typically after promoter induction, although expressioncan be constitutive, the proteins may form insoluble aggregates. Thereare several protocols that are suitable for purification of proteininclusion bodies. For example, purification of aggregate proteins(hereinafter referred to as inclusion bodies) typically involves theextraction, separation and/or purification of inclusion bodies bydisruption of bacterial cells typically, but not limited to, byincubation in a buffer of about 100-150 μg/ml lysozyme and 0.1% NonidetP40, a non-ionic detergent. The cell suspension can be ground using aPolytron grinder (Brinkman Instruments, Westbury, N.Y.). Alternatively,the cells can be sonicated on ice. Alternate methods of lysing bacteriaare described in Ausubel et al. and Sambrook et al., both supra, andwill be apparent to those of skill in the art.

The cell suspension is generally centrifuged and the pellet containingthe inclusion bodies resuspended in buffer which does not dissolve butwashes the inclusion bodies, e.g., 20 mM Tris-HCl (pH 7.2), 1 mM EDTA,150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. It may benecessary to repeat the wash step to remove as much cellular debris aspossible. The remaining pellet of inclusion bodies may be resuspended inan appropriate buffer (e.g., 20 mM sodium phosphate, pH 6.8, 150 mMNaCl). Other appropriate buffers will be apparent to those of skill inthe art.

Following the washing step, the inclusion bodies are solubilized by theaddition of a solvent that is both a strong hydrogen acceptor and astrong hydrogen donor (or a combination of solvents each having one ofthese properties). The proteins that formed the inclusion bodies maythen be renatured by dilution or dialysis with a compatible buffer.Suitable solvents include, but are not limited to, urea (from about 4 Mto about 8 M), formamide (at least about 80%, volume/volume basis), andguanidine hydrochloride (from about 4 M to about 8 M). Some solventsthat are capable of solubilizing aggregate-forming proteins, such as SDS(sodium dodecyl sulfate) and 70% formic acid, are inappropriate for usein this procedure due to the possibility of irreversible denaturation ofthe proteins, accompanied by a lack of immunogenicity and/or activity.Although guanidine hydrochloride and similar agents are denaturants,this denaturation is not irreversible and renaturation may occur uponremoval (by dialysis, for example) or dilution of the denaturant,allowing re-formation of the immunologically and/or biologically activeprotein of interest. After solubilization, the protein can be separatedfrom other bacterial proteins by standard separation techniques.

Alternatively, it is possible to purify proteins from bacteriaperiplasm. Where the protein is exported into the periplasm of thebacteria, the periplasmic fraction of the bacteria can be isolated bycold osmotic shock in addition to other methods known to those of skillin the art (see, Ausubel et al., supra). To isolate recombinant proteinsfrom the periplasm, the bacterial cells are centrifuged to form apellet. The pellet is resuspended in a buffer containing 20% sucrose. Tolyse the cells, the bacteria are centrifuged and the pellet isresuspended in ice-cold 5 mM MgSO₄ and kept in an ice bath forapproximately 10 minutes. The cell suspension is centrifuged and thesupernatant decanted and saved. The recombinant proteins present in thesupernatant can be separated from the host proteins by standardseparation techniques well known to those of skill in the art.

B. Purification of Proteins from Insect Cells

Proteins can also be purified from eukaryotic gene expression systems asdescribed in, e.g., Fernandez and Hoeffler, Gene Expression Systems(1999). In some embodiments, baculovirus expression systems are used toisolate proteins of the invention. Recombinant baculoviruses aregenerally generated by replacing the polyhedrin coding sequence of abaculovirus with a gene to be expressed (e.g., encoding a polypeptide ofthe invention). Viruses lacking the polyhedrin gene have a unique plaquemorphology making them easy to recognize. In some embodiments, arecombinant baculovirus is generated by first cloning a polynucleotideof interest into a transfer vector (e.g., a pUC based vector) such thatthe polynucleotide is operably linked to a polyhedrin promoter. Thetransfer vector is transfected with wildtype DNA into an insect cell(e.g., Sf9, Sf21 or BT1-TN-5B1-4 cells), resulting in homologousrecombination and replacement of the polyhedrin gene in the wildtypeviral DNA with the polynucleotide of interest. Virus can then begenerated and plaque purified. Protein expression results upon viralinfection of insect cells. Expressed proteins can be harvested from cellsupernatant if secreted, or from cell lysates if intracellular. See,e.g., Ausubel et al. and Fernandez and Hoeffler, supra.

C. Standard Protein Separation Techniques for Purifying Proteins

1. Solubility Fractionation

Often as an initial step, and if the protein mixture is complex, aninitial salt fractionation can separate many of the unwanted host cellproteins (or proteins derived from the cell culture media) from therecombinant protein of interest. The preferred salt is ammonium sulfate.Ammonium sulfate precipitates proteins by effectively reducing theamount of water in the protein mixture. Proteins then precipitate on thebasis of their solubility. The more hydrophobic a protein is, the morelikely it is to precipitate at lower ammonium sulfate concentrations. Atypical protocol is to add saturated ammonium sulfate to a proteinsolution so that the resultant ammonium sulfate concentration is between20-30%. This will precipitate the most hydrophobic proteins. Theprecipitate is discarded (unless the protein of interest is hydrophobic)and ammonium sulfate is added to the supernatant to a concentrationknown to precipitate the protein of interest. The precipitate is thensolubilized in buffer and the excess salt removed if necessary, througheither dialysis or diafiltration. Other methods that rely on solubilityof proteins, such as cold ethanol precipitation, are well known to thoseof skill in the art and can be used to fractionate complex proteinmixtures.

2. Size Differential Filtration

Based on a calculated molecular weight, a protein of greater and lessersize can be isolated using ultrafiltration through membranes ofdifferent pore sizes (for example, Amicon or Millipore membranes). As afirst step, the protein mixture is ultrafiltered through a membrane witha pore size that has a lower molecular weight cut-off than the molecularweight of the protein of interest. The retentate of the ultrafiltrationis then ultrafiltered against a membrane with a molecular cut offgreater than the molecular weight of the protein of interest. Therecombinant protein will pass through the membrane into the filtrate.The filtrate can then be chromatographed as described below.

3. Column Chromatography

The proteins of interest can also be separated from other proteins onthe basis of their size, net surface charge, hydrophobicity and affinityfor ligands. In addition, antibodies raised against proteins can beconjugated to column matrices and the proteins immunopurified. All ofthese methods are well known in the art.

Immunoaffinity chromatography using antibodies raised to a variety ofaffinity tags such as hemagglutinin (HA), FLAG, Xpress, Myc,hexahistidine (SEQ ID NO:41) (His), glutathione S transferase (GST) andthe like can be used to purify polypeptides. The His tag will also actas a chelating agent for certain metals (e.g., Ni) and thus the metalscan also be used to purify His-containing polypeptides. Afterpurification, the tag is optionally removed by specific proteolyticcleavage.

It will be apparent to one of skill that chromatographic techniques canbe performed at any scale and using equipment from many differentmanufacturers (e.g., Pharmacia Biotech).

V. Detection of Polynucleotides of the Invention

Those of skill in the art will recognize that detection of expression ofpolynucleotides and polypeptides of the invention has many uses. Forexample, as discussed herein, detection of levels of polynucleotides andpolypeptides of the invention in a patient is useful for diagnosingdiabetes or a predisposition for at least some of the pathologicaleffects of diabetes. In addition, at least some familial diabetes can bediagnosed by detecting the IC-RFX allele in an individual. Moreover,detection of gene expression is useful to identify modulators ofexpression of polynucleotides and polypeptides of the invention.

A variety of methods of specific DNA and RNA measurement that usenucleic acid hybridization techniques are known to those of skill in theart (see, Sambrook, supra). Some methods involve an electrophoreticseparation (e.g., Southern blot for detecting DNA, and Northern blot fordetecting RNA), but measurement of DNA and RNA can also be carried outin the absence of electrophoretic separation (e.g., by dot blot).Southern blot of genomic DNA (e.g., from a human) can be used forscreening for restriction fragment length polymorphism (RFLP) to detectthe presence of a genetic disorder affecting a polypeptide of theinvention. In some cases, a polynucleotide from a subject is isolated(e.g., by PCR amplification) and sequenced to detect mutations (e.g.,deletions, insertions, point mutations).

The selection of a nucleic acid hybridization format is not critical. Avariety of nucleic acid hybridization formats are known to those skilledin the art. For example, common formats include sandwich assays andcompetition or displacement assays. Hybridization techniques aregenerally described in Hames and Higgins Nucleic Acid Hybridization, APractical Approach, IRL Press (1985); Gall and Pardue, Proc. Natl. Acad.Sci. U.S.A., 63:378-383 (1969); and John et al. Nature, 223:582-587(1969).

Detection of a hybridization complex may require the binding of asignal-generating complex to a duplex of target and probepolynucleotides or nucleic acids. Typically, such binding occurs throughligand and anti-ligand interactions as between a ligand-conjugated probeand an anti-ligand conjugated with a signal. The binding of the signalgeneration complex is also readily amenable to accelerations by exposureto ultrasonic energy.

The label may also allow indirect detection of the hybridizationcomplex. For example, where the label is a hapten or antigen, the samplecan be detected by using antibodies. In these systems, a signal isgenerated by attaching fluorescent or enzyme molecules to the antibodiesor in some cases, by attachment to a radioactive label (see, e.g.,Tijssen, “Practice and Theory of Enzyme Immunoassays,” LaboratoryTechniques in Biochemistry and Molecular Biology, Burdon and vanKnippenberg Eds., Elsevier (1985), pp. 9-20).

The probes are typically labeled either directly, as with isotopes,chromophores, lumiphores, chromogens, or indirectly, such as withbiotin, to which a streptavidin complex may later bind. Thus, thedetectable labels used in the assays of the present invention can beprimary labels (where the label comprises an element that is detecteddirectly or that produces a directly detectable element) or secondarylabels (where the detected label binds to a primary label, e.g., as iscommon in immunological labeling). Typically, labeled signal nucleicacids are used to detect hybridization. Complementary nucleic acids orsignal nucleic acids may be labeled by any one of several methodstypically used to detect the presence of hybridized polynucleotides. Themost common method of detection is the use of autoradiography with ³H,¹²⁵I, ³⁵S, ¹⁴C, or ³²P-labeled probes or the like.

Other labels include, e.g., ligands that bind to labeled antibodies,fluorophores, chemiluminescent agents, enzymes, and antibodies that canserve as specific binding pair members for a labeled ligand. Anintroduction to labels, labeling procedures and detection of labels isfound in Polak and Van Noorden Introduction to Immunocytochemistry, 2nded., Springer Verlag, NY (1997); and in Haugland Handbook of FluorescentProbes and Research Chemicals, a combined handbook and cataloguePublished by Molecular Probes, Inc. (1996).

In general, a detector that monitors a particular probe or probecombination is used to detect the detection reagent label. Typicaldetectors include spectrophotometers, phototubes and photodiodes,microscopes, scintillation counters, cameras, film and the like, as wellas combinations thereof. Examples of suitable detectors are widelyavailable from a variety of commercial sources known to persons of skillin the art. Commonly, an optical image of a substrate comprising boundlabeling moieties is digitized for subsequent computer analysis.

The amount of, for example, an RNA is measured by quantifying the amountof label fixed to the solid support by binding of the detection reagent.Typically, the presence of a modulator during incubation will increaseor decrease the amount of label fixed to the solid support relative to acontrol incubation that does not comprise the modulator, or as comparedto a baseline established for a particular reaction type. Means ofdetecting and quantitating labels are well known to those of skill inthe art.

In some embodiments, the target nucleic acid or the probe is immobilizedon a solid support. Solid supports suitable for use in the assays of theinvention are known to those of skill in the art. As used herein, asolid support is a matrix of material in a substantially fixedarrangement.

A variety of automated solid-phase assay techniques are alsoappropriate. For instance, very large scale immobilized polymer arrays(VLSIPS™), i.e. Gene Chips or microarrays, available from Affymetrix,Inc. in Santa Clara, Calif. can be used to detect changes in expressionlevels of a plurality of genes involved in the same regulatory pathwayssimultaneously. See, Tijssen, supra., Fodor et al. (1991) Science, 251:767-777; Sheldon et al. (1993) Clinical Chemistry 39(4): 718-719, andKozal et al. (1996) Nature Medicine 2(7): 753-759. Similarly, spottedcDNA arrays (arrays of cDNA sequences bound to nylon, glass or anothersolid support) can also be used to monitor expression of a plurality ofgenes.

Typically, the array elements are organized in an ordered fashion sothat each element is present at a specified location on the substrate.Because the array elements are at specified locations on the substrate,the hybridization patterns and intensities (which together create aunique expression profile) can be interpreted in terms of expressionlevels of particular genes and can be correlated with a particulardisease or condition or treatment. See, e.g., Schena et al., Science270: 467-470 (1995) and Lockhart et al., Nature Biotech. 14: 1675-1680(1996).

Detection of nucleic acids can also be accomplished, for example, byusing a labeled detection moiety that binds specifically to duplexnucleic acids (e.g., an antibody that is specific for RNA-DNA duplexes).One example uses an antibody that recognizes DNA-RNA heteroduplexes inwhich the antibody is linked to an enzyme (typically by recombinant orcovalent chemical bonding). The antibody is detected when the enzymereacts with its substrate, producing a detectable product. Coutlee etal. (1989) Analytical Biochemistry 181:153-162; Bogulavski (1986) et al.J. Immunol. Methods 89:123-130; Prooijen-Knegt (1982) Exp. Cell Res.141:397-407; Rudkin (1976) Nature 265:472-473, Stollar (1970) PNAS65:993-1000; Ballard (1982) Mol. Immunol. 19:793-799; Pisetsky andCaster (1982) Mol. Immunol. 19:645-650; Viscidi et al. (1988) J. Clin.Microbial. 41:199-209; and Kiney et al. (1989) J. Clin. Microbiol.27:6-12 describe antibodies to RNA duplexes, including homo andheteroduplexes. Kits comprising antibodies specific for DNA:RNA hybridsare available, e.g., from Digene Diagnostics, Inc. (Beltsville, Md.).

In addition to available antibodies, one of skill in the art can easilymake antibodies specific for nucleic acid duplexes using existingtechniques, or modify those antibodies that are commercially or publiclyavailable. In addition to the art referenced above, general methods forproducing polyclonal and monoclonal antibodies are known to those ofskill in the art (see, e.g., Paul (ed) Fundamental Immunology, ThirdEdition Raven Press, Ltd., NY (1993); Coligan Current Protocols inImmunology Wiley/Greene, NY (1991); Harlow and Lane Antibodies: ALaboratory Manual Cold Spring Harbor Press, NY (1989); Stites et al.(eds.) Basic and Clinical Immunology (4th ed.) Lange MedicalPublications, Los Altos, Calif., and references cited therein; GodingMonoclonal Antibodies: Principles and Practice (2d ed.) Academic Press,New York, N.Y., (1986); and Kohler and Milstein Nature 256: 495-497(1975)). Other suitable techniques for antibody preparation includeselection of libraries of recombinant antibodies in phage or similarvectors (see, Huse et al. Science 246:1275-1281 (1989); and Ward et al.Nature 341:544-546 (1989)). Specific monoclonal and polyclonalantibodies and antisera will usually bind with a K_(D) of at least about0.1 μM, preferably at least about 0.01 μM or better, and most typicallyand preferably, 0.001 μM or better.

The nucleic acids used in this invention can be either positive ornegative probes. Positive probes bind to their targets and the presenceof duplex formation is evidence of the presence of the target. Negativeprobes fail to bind to the suspect target and the absence of duplexformation is evidence of the presence of the target. For example, theuse of a wild type specific nucleic acid probe or PCR primers may serveas a negative probe in an assay sample where only the nucleotidesequence of interest is present.

The sensitivity of the hybridization assays may be enhanced through useof a nucleic acid amplification system that multiplies the targetnucleic acid being detected. Examples of such systems include thepolymerase chain reaction (PCR) system and the ligase chain reaction(LCR) system. Other methods recently described in the art are thenucleic acid sequence based amplification (NASBA, Cangene, Mississauga,Ontario) and Q Beta Replicase systems. These systems can be used todirectly identify mutants where the PCR or LCR primers are designed tobe extended or ligated only when a selected sequence is present.Alternatively, the selected sequences can be generally amplified using,for example, nonspecific PCR primers and the amplified target regionlater probed for a specific sequence indicative of a mutation. It isunderstood that various detection probes, including Taqman and molecularbeacon probes can be used to monitor amplification reaction products,e.g., in real time.

An alternative means for determining the level of expression of thenucleic acids of the present invention is in situ hybridization. In situhybridization assays are well known and are generally described inAngerer et al., Methods Enzymol. 152:649-660 (1987). In an in situhybridization assay, cells, preferentially human cells from thecerebellum or the hippocampus, are fixed to a solid support, typically aglass slide. If DNA is to be probed, the cells are denatured with heator alkali. The cells are then contacted with a hybridization solution ata moderate temperature to permit annealing of specific probes that arelabeled. The probes are preferably labeled with radioisotopes orfluorescent reporters.

VI. Immunological Detection of Polypeptides of the Invention

In addition to the detection of IC-RFX polynucleotides of the inventionand gene expression using nucleic acid hybridization technology, one canalso use immunoassays to detect polypeptides of the invention.Immunoassays can be used to qualitatively or quantitatively analyzepolypeptides of the invention. A general overview of the applicabletechnology can be found in Harlow & Lane, Antibodies: A LaboratoryManual (1988).

A. Antibodies to Target Proteins or Other Immunogens

Methods for producing polyclonal and monoclonal antibodies that reactspecifically with a protein of interest or other immunogen are known tothose of skill in the art (see, e.g., Coligan, supra; and Harlow andLane, supra; Stites et al., supra and references cited therein; Goding,supra; and Kohler and Milstein Nature, 256:495-497 (1975)). Suchtechniques include antibody preparation by selection of antibodies fromlibraries of recombinant antibodies in phage or similar vectors (see,Huse et al., supra; and Ward et al., supra). For example, in order toproduce antisera for use in an immunoassay, the protein of interest oran antigenic fragment thereof, is isolated as described herein. Forexample, a recombinant protein is produced in a transformed cell line.An inbred strain of mice or rabbits is immunized with the protein usinga standard adjuvant, such as Freund's adjuvant, and a standardimmunization protocol. Alternatively, a synthetic peptide derived fromthe sequences disclosed herein is conjugated to a carrier protein andused as an immunogen.

Polyclonal sera are collected and titered against the immunogen in animmunoassay, for example, a solid phase immunoassay with the immunogenimmobilized on a solid support. Polyclonal antisera with a titer of 10⁴or greater are selected and tested for their crossreactivity againstproteins other than the polypeptides of the invention or even otherhomologous proteins from other organisms, using a competitive bindingimmunoassay. Specific monoclonal and polyclonal antibodies and antiserawill usually bind with a K_(D) of at least about 0.1 mM, more usually atleast about 1 μM, preferably at least about 0.1 μM or better, and mostpreferably, 0.01 μM or better.

A number of proteins of the invention comprising immunogens may be usedto produce antibodies specifically or selectively reactive with theproteins of interest. Recombinant protein is the preferred immunogen forthe production of monoclonal or polyclonal antibodies. Naturallyoccurring protein may also be used either in pure or impure form.Synthetic peptides made using the protein sequences described herein mayalso be used as an immunogen for the production of antibodies to theprotein. Recombinant protein can be expressed in eukaryotic orprokaryotic cells and purified as generally described supra. The productis then injected into an animal capable of producing antibodies. Eithermonoclonal or polyclonal antibodies may be generated for subsequent usein immunoassays to measure the protein.

Methods of production of polyclonal antibodies are known to those ofskill in the art. In brief, an immunogen, preferably a purified protein,is mixed with an adjuvant and animals are immunized. The animal's immuneresponse to the immunogen preparation is monitored by taking test bleedsand determining the titer of reactivity to polypeptides of theinvention. When appropriately high titers of antibody to the immunogenare obtained, blood is collected from the animal and antisera areprepared. Further fractionation of the antisera to enrich for antibodiesreactive to the protein can be done if desired (see, Harlow and Lane,supra).

Monoclonal antibodies may be obtained using various techniques familiarto those of skill in the art. Typically, spleen cells from an animalimmunized with a desired antigen are immortalized, commonly by fusionwith a myeloma cell (see, Kohler and Milstein, Eur. J. Immunol.6:511-519 (1976)). Alternative methods of immortalization include, e.g.,transformation with Epstein Barr Virus, oncogenes, or retroviruses, orother methods well known in the art. Colonies arising from singleimmortalized cells are screened for production of antibodies of thedesired specificity and affinity for the antigen, and yield of themonoclonal antibodies produced by such cells may be enhanced by varioustechniques, including injection into the peritoneal cavity of avertebrate host. Alternatively, one may isolate DNA sequences thatencode a monoclonal antibody or a binding fragment thereof by screeninga DNA library from human B cells according to the general protocoloutlined by Huse et al., supra.

Once target immunogen-specific antibodies are available, the immunogencan be measured by a variety of immunoassay methods with qualitative andquantitative results available to the clinician. For a review ofimmunological and immunoassay procedures in general see, Stites, supra.Moreover, the immunoassays of the present invention can be performed inany of several configurations, which are reviewed extensively in MaggioEnzyme Immunoassay, CRC Press, Boca Raton, Fla. (1980); Tijssen, supra;and Harlow and Lane, supra.

Immunoassays to measure target proteins in a human sample may use apolyclonal antiserum that was raised to full-length polypeptides of theinvention or a fragment thereof. This antiserum is selected to have lowcross-reactivity against other proteins and any such cross-reactivity isremoved by immunoabsorption prior to use in the immunoassay.

B. Immunological Binding Assays

In some embodiments, a protein of interest is detected and/or quantifiedusing any of a number of well-known immunological binding assays (see,e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168).For a review of the general immunoassays, see also Asai Methods in CellBiology Volume 37.: Antibodies in Cell Biology, Academic Press, Inc. NY(1993); Stites, supra. Immunological binding assays (or immunoassays)typically utilize a “capture agent” to specifically bind to and oftenimmobilize the analyte (e.g., full-length polypeptides of the presentinvention, or antigenic subsequences thereof). The capture agent is amoiety that specifically binds to the analyte. The antibody may beproduced by any of a number of means well known to those of skill in theart and as described above.

Immunoassays also often utilize a labeling agent to bind specifically toand label the binding complex formed by the capture agent and theanalyte. The labeling agent may itself be one of the moieties comprisingthe antibody/analyte complex. Alternatively, the labeling agent may be athird moiety, such as another antibody, that specifically binds to theantibody/protein complex.

In a preferred embodiment, the labeling agent is a second antibodybearing a label. Alternatively, the second antibody may lack a label,but it may, in turn, be bound by a labeled third antibody specific toantibodies of the species from which the second antibody is derived. Thesecond antibody can be modified with a detectable moiety, such asbiotin, to which a third labeled molecule can specifically bind, such asenzyme-labeled streptavidin.

Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G, can also be used as the labelagents. These proteins are normal constituents of the cell walls ofstreptococcal bacteria. They exhibit a strong non-immunogenic reactivitywith immunoglobulin constant regions from a variety of species (see,generally, Kronval, et al. J. Immunol., 111: 1401-1406 (1973); andAkerstrom, et al. J. Immunol., 135:2589-2542 (1985)).

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. The incubation time will depend upon the assay format, analyte,volume of solution, concentrations, and the like. Usually, the assayswill be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 10° C. to 40° C.

1. Non-Competitive Assay Formats

Immunoassays for detecting proteins or analytes of interest from tissuesamples may be either competitive or noncompetitive. Noncompetitiveimmunoassays are assays in which the amount of captured protein oranalyte is directly measured. In one preferred “sandwich” assay, forexample, the capture agent (e.g., antibodies specific for thepolypeptides of the invention) can be bound directly to a solidsubstrate where it is immobilized. These immobilized antibodies thencapture the polypeptide present in the test sample. The polypeptide ofthe invention thus immobilized is then bound by a labeling agent, suchas a second labelled antibody specific for the polypeptide.Alternatively, the second antibody may lack a label, but it may, inturn, be bound by a labeled third antibody specific to antibodies of thespecies from which the second antibody is derived. The second can bemodified with a detectable moiety, such as biotin, to which a thirdlabeled molecule can specifically bind, such as enzyme-labeledstreptavidin.

2. Competitive Assay Formats

In competitive assays, the amount of protein or analyte present in thesample is measured indirectly by measuring the amount of an added(exogenous) protein or analyte displaced (or competed away) from aspecific capture agent (e.g., antibodies specific for a polypeptide ofthe invention) by the protein or analyte present in the sample. Theamount of immunogen bound to the antibody is inversely proportional tothe concentration of immunogen present in the sample. In a particularlypreferred embodiment, the antibody is immobilized on a solid substrate.The amount of analyte may be detected by providing a labeled analytemolecule. It is understood that labels can include, e.g., radioactivelabels as well as peptide or other tags that can be recognized bydetection reagents such as antibodies.

Immunoassays in the competitive binding format can be used forcross-reactivity determinations. For example, the protein encoded by thesequences described herein can be immobilized on a solid support.Proteins are added to the assay and compete with the binding of theantisera to the immobilized antigen. The ability of the above proteinsto compete with the binding of the antisera to the immobilized proteinis compared to that of the protein encoded by any of the sequencesdescribed herein. The percent cross-reactivity for the above proteins iscalculated, using standard calculations. Those antisera with less than10% cross-reactivity with each of the proteins listed above are selectedand pooled. The cross-reacting antibodies are optionally removed fromthe pooled antisera by immunoabsorption with the considered proteins,e.g., distantly related homologs.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein,thought to be perhaps a protein of the present invention, to theimmunogen protein. In order to make this comparison, the two proteinsare each assayed at a wide range of concentrations and the amount ofeach protein required to inhibit 50% of the binding of the antisera tothe immobilized protein is determined. If the amount of the secondprotein required is less than 10 times the amount of the proteinpartially encoded by a sequence herein that is required, then the secondprotein is said to specifically bind to an antibody generated to animmunogen consisting of the target protein.

3. Other Assay Formats

In some embodiments, western blot (immunoblot) analysis is used todetect and quantify the presence of a polypeptide of the invention inthe sample. The technique generally comprises separating sample proteinsby gel electrophoresis on the basis of molecular weight, transferringthe separated proteins to a suitable solid support (such as, e.g., anitrocellulose filter, a nylon filter, or a derivatized nylon filter)and incubating the sample with the antibodies that specifically bind theprotein of interest. For example, antibodies are selected thatspecifically bind to the polypeptides of the invention on the solidsupport. These antibodies may be directly labeled or alternatively maybe subsequently detected using labeled antibodies (e.g., labeled sheepanti-mouse antibodies) that specifically bind to the antibodies againstthe protein of interest.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see, Monroe et al.(1986) Amer. Clin. Prod. Rev. 5:34-41).

4. Labels

The particular label or detectable group used in the assay is not acritical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most labels useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., Dynabeads™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and calorimetric labels such ascolloidal gold or colored glass or plastic (e.g., polystyrene,polypropylene, latex, etc.) beads.

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on the sensitivity required, the ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Themolecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorescent compound.A variety of enzymes and fluorescent compounds can be used with themethods of the present invention and are well-known to those of skill inthe art (for a review of various labeling or signal producing systemswhich may be used, see, e.g., U.S. Pat. No. 4,391,904).

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple calorimetriclabels may be detected directly by observing the color associated withthe label. Thus, in various dipstick assays, conjugated gold oftenappears pink, while various conjugated beads appear the color of thebead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need to be labeled and the presence ofthe target antibody is detected by simple visual inspection.

VII. Identification of Modulators of Polypeptides of the Invention

Modulators of a IC-RFX polypeptide of the invention, i.e. agonists orantagonists of an IC-RFX polypeptide's activity, or an IC-RFXpolypeptide's or an IC-RFX polynucleotide's expression, are useful fortreating a number of human diseases, including diabetes. For example,administration of modulators can be used to treat diabetic patients orprediabetic individuals to prevent progression, and therefore symptoms,associated with diabetes.

A. Agents that Modulate Polypeptides of the Invention

The agents tested as modulators of polypeptides of the invention can beany small chemical compound, or a biological entity, such as a protein,sugar, nucleic acid or lipid. Typically, test compounds will be smallchemical molecules and peptides. Essentially any chemical compound canbe used as a potential modulator or ligand in the assays of theinvention, although most often compounds that can be dissolved inaqueous or organic (especially DMSO-based) solutions are used. Theassays are designed to screen large chemical libraries by automating theassay steps and providing compounds from any convenient source toassays, which are typically run in parallel (e.g., in microtiter formatson microtiter plates in robotic assays). Modulators also include agentsdesigned to reduce the level of mRNA encoding a polypeptide of theinvention (e.g. antisense molecules, ribozymes, DNAzymes, smallinhibitory RNAs and the like) or the level of translation from an mRNA(e.g., translation blockers such as an antisense molecules that arecomplementary to translation start or other sequences on an mRNAmolecule). It will be appreciated that there are many suppliers ofchemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St.Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-BiochemicaAnalytika (Buchs, Switzerland) and the like.

In some embodiments, high throughput screening methods involve providinga combinatorial chemical or peptide library containing a large number ofpotential therapeutic compounds (potential modulator compounds). Such“combinatorial chemical libraries” or “ligand libraries” are thenscreened in one or more assays, as described herein, to identify thoselibrary members (particular chemical species or subclasses) that displaya desired characteristic activity. The compounds thus identified canserve as conventional “lead compounds” or can themselves be used aspotential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091),benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat.Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagiharaet al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314(1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang etal., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, 5,288,514, and thelike).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Tripos, Inc., St. Louis, Mo., 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

B. Methods of Screening for Modulators of the Polypeptides of theInvention

A number of different screening protocols can be utilized to identifyagents that modulate the level of expression or activity of apolynucleotide of a polypeptide of the invention in cells, particularlymammalian cells, and especially human cells. In general terms, thescreening methods involve screening a plurality of agents to identify anagent that modulates the activity of a polypeptide of the invention by,e.g., binding to the polypeptide, preventing an inhibitor or activatorfrom binding to the polypeptide, increasing association of an inhibitoror activator with the polypeptide, or activating or inhibitingexpression of the polypeptide.

Any cell expressing a full-length polypeptide of the invention or afragment thereof can be used to identify modulators. In someembodiments, the cells are eukaryotic cells lines (e.g., CHO or HEK293)transformed to express a heterologous polypeptide of the invention. Insome embodiments, a cell expressing an endogenous polypeptide of theinvention is used in screens. In other embodiments, modulators arescreened for their ability to effect insulin production of a cell or toinduce other β-cell phenotypes in a cell.

1. Polypeptide Binding Assays

Preliminary screens can be conducted by screening for agents capable ofbinding to polypeptides of the invention, as at least some of the agentsso identified are likely modulators of a polypeptide of the invention.Binding assays are also useful, e.g., for identifying endogenousproteins that interact with polypeptides of the invention. For example,antibodies, receptors or other molecules that bind polypeptides of theinvention can be identified in binding assays.

Binding assays usually involve contacting a polypeptide of the inventionwith one or more test agents and allowing sufficient time for theprotein and test agents to form a binding complex. Any binding complexesformed can be detected using any of a number of established analyticaltechniques. Protein binding assays include, but are not limited to,methods that measure co-precipitation or co-migration on non-denaturingSDS-polyacrylamide gels, and co-migration on Western blots (see, e.g.,Bennet, J. P. and Yamamura, H. I. (1985) “Neurotransmitter, Hormone orDrug Receptor Binding Methods,” in Neurotransmitter Receptor Binding(Yamamura, H. I., et al., eds.), pp. 61-89). Other binding assaysinvolve the use of mass spectrometry or NMR techniques to identifymolecules bound to a polypeptide of the invention or displacement oflabeled substrates. The polypeptides of the invention utilized in suchassays can be naturally expressed, cloned or synthesized.

In addition, mammalian or yeast two-hybrid approaches (see, e.g.,Bartel, P. L. et. al. Methods Enzymol, 254:241 (1995)) can be used toidentify polypeptides or other molecules that interact or bind whenexpressed together in a host cell.

2. Polypeptide Activity

The activity of polypeptides of the invention can be assessed using avariety of in vitro and in vivo assays to determine functional,chemical, and physical effects, e.g., measuring ligand binding (e.g.,radioactive or otherwise labeled ligand binding), second messengers(e.g., cAMP, cGMP, IP₃, DAG, or Ca²⁺), ion flux, phosphorylation levels,transcription levels, and the like. Furthermore, such assays can be usedto test for inhibitors and activators of the polypeptides of theinvention. Modulators can also be genetically altered versions ofpolypeptides of the invention.

The polypeptide of the assay will be selected from a polypeptide withsubstantial identity to a sequence of SEQ ID NO:2, or otherconservatively modified variants thereof. Generally, the amino acidsequence identity will be at least 70%, optionally at least 85%,optionally at least 90-95% to the polypeptides exemplified herein.Optionally, the polypeptide of the assays will comprise a fragment of apolypeptide of the invention, such as an extracellular domain,transmembrane domain, cytoplasmic domain, ligand binding domain, subunitassociation domain, active site, and the like. Either a polypeptide ofthe invention or a domain thereof can be covalently linked to aheterologous protein to create a chimeric protein used in the assaysdescribed herein.

Modulators of polypeptide activity are tested using either recombinantor naturally occurring polypeptides of the invention. The protein can beisolated, expressed in a cell, expressed in a membrane derived from acell, expressed in tissue or in an animal, either recombinant ornaturally occurring. For example, tissue slices, dissociated cells,e.g., from tissues expressing polypeptides of the invention, transformedcells, or membranes can be used. Modulation is tested using one of thein vitro or in vivo assays described herein.

Modulator binding to polypeptides of the invention, a domain, orchimeric protein can be tested in solution, in a bilayer membrane,attached to a solid phase, in a lipid monolayer, or in vesicles. Bindingof a modulator can be tested using, e.g., changes in spectroscopiccharacteristics (e.g., fluorescence, absorbance, refractive index),hydrodynamic (e.g., shape), chromatographic, or solubility properties.

Samples or assays that are treated with a potential modulator (e.g., a“test compound”) are compared to control samples without the testcompound, to examine the extent of modulation. Control samples(untreated with activators or inhibitors) are assigned a relativeactivity value of 100. Inhibition of the polypeptides of the inventionis achieved when the activity value relative to the control is about90%, optionally 50%, optionally 25-0%. Activation of the polypeptides ofthe invention is achieved when the activity value relative to thecontrol is 110%, optionally 150%, 200%, 300%, 400%, 500%, or 1000-2000%.

3. Expression Assays

Screening for a compound that modulates the expression of apolynucleotide or a polypeptide of the invention is also provided.Screening methods generally involve conducting cell-based assays inwhich test compounds are contacted with one or more cells expressing apolynucleotide or a polypeptide of the invention, and then detecting anincrease or decrease in expression (either transcript or translationproduct). Assays can be performed with any cells that express apolynucleotide or a polypeptide of the invention.

Expression can be detected in a number of different ways. As describedinfra, the expression level of a polynucleotide of the invention in acell can be determined by probing the mRNA expressed in a cell with aprobe that specifically hybridizes with a transcript (or complementarynucleic acid derived therefrom) of a polynucleotide of the invention.Probing can be conducted by lysing the cells and conducting Northernblots or without lysing the cells using in situ-hybridizationtechniques. Alternatively, a polypeptide of the invention can bedetected using immunological methods in which a cell lysate is probedwith antibodies that specifically bind to the polypeptide.

The level of expression or activity of a polynucleotide or a polypeptideof the invention can be compared to a baseline value. The baseline valuecan be a value for a control sample or a statistical value that isrepresentative of expression levels of a polynucleotide or a polypeptideof the invention for a control population (e.g., lean non-diabeticindividuals) or cells (e.g., normal β-cells that produce insulin). As anegative control, expression levels can also be determined for cellsthat do not express the polynucleotide or a polypeptide of theinvention. Such cells generally are otherwise substantially geneticallyidentical to the test cells.

A variety of different types of cells can be utilized in the reporterassays. Cells that do not endogenously express a polypeptide of theinvention can be prokaryotic, but are preferably eukaryotic. Theeukaryotic cells can be any of the cells typically utilized ingenerating cells that harbor recombinant nucleic acid constructs.Exemplary eukaryotic cells include, but are not limited to, yeast, andvarious higher eukaryotic cells such as the HEK293, HepG2, COS, CHO andHeLa cell lines.

Various controls can be conducted to ensure that an observed activity isauthentic including running parallel reactions with cells that lack thereporter construct or by not contacting a cell harboring the reporterconstruct with test compound. Compounds can also be further validated asdescribed below.

4. Validation

Agents that are initially identified by any of the foregoing screeningmethods can be further tested to validate the apparent activity.Modulators that are selected for further study can be tested for aneffect on insulin levels in animals.

For example, the effect of the compound can be assessed in diabeticanimals. The blood glucose and insulin levels can be determined. Theanimal models utilized in validation studies generally are mammals ofany kind. Specific examples of suitable animals include, but are notlimited to, primates, mice and rats. For example, monogenic models ofdiabetes (e.g., ob/ob and db/db mice, Zucker rats and Zucker DiabeticFatty rats etc) or polygenic models of diabetes (e.g., OLETF rats, GKrats, NSY mice, and KK mice) can be useful for validating modulation ofa polypeptide of the invention in a diabetic or insulin resistantanimal. In addition, transgenic animals expressing human polypeptides ofthe invention can be used to further validate drug candidates.

C. Solid Phase and Soluble High Throughput Assays

In the high throughput assays of the invention, it is possible to screenup to several thousand different modulators or ligands in a single day.In particular, each well of a microtiter plate can be used to run aseparate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 100 (e.g., 96) modulators. If 1536 well plates areused, then a single plate can easily assay from about 100 to about 1500different compounds. It is possible to assay several different platesper day; assay screens for up to about 6,000-20,000 or more differentcompounds are possible using the integrated systems of the invention. Inaddition, microfluidic approaches to reagent manipulation can be used.

A molecule of interest (e.g., a polypeptide or polynucleotide of theinvention, or a modulator thereof) can be bound to the solid-statecomponent, directly or indirectly, via covalent or non-covalent linkage,e.g., via a tag. The tag can be any of a variety of components. Ingeneral, a molecule that binds the tag (a tag binder) is fixed to asolid support, and the tagged molecule of interest is attached to thesolid support by interaction of the tag and the tag binder.

A number of tags and tag binders can be used, based upon known molecularinteractions well described in the literature. For example, where a taghas a natural binder, for example, biotin, protein A, or protein G, itcan be used in conjunction with appropriate tag binders (avidin,streptavidin, neutravidin, the Fc region of an immunoglobulin, poly-His,etc.) Antibodies to molecules with natural binders such as biotin arealso widely available and appropriate tag binders (see, SIGMAImmunochemicals 1998 catalogue SIGMA, St. Louis Mo.).

Similarly, any haptenic or antigenic compound can be used in combinationwith an appropriate antibody to form a tag/tag binder pair. Thousands ofspecific antibodies are commercially available and many additionalantibodies are described in the literature. For example, in one commonconfiguration, the tag is a first antibody and the tag binder is asecond antibody that recognizes the first antibody. In addition toantibody-antigen interactions, receptor-ligand interactions are alsoappropriate as tag and tag-binder pairs, such as agonists andantagonists of cell membrane receptors (e.g., cell receptor-ligandinteractions such as transferrin, c-kit, viral receptor ligands,cytokine receptors, chemokine receptors, interleukin receptors,immunoglobulin receptors and antibodies, the cadherin family, theintegrin family, the selectin family, and the like; see, e.g., Pigott &Power, The Adhesion Molecule Facts Book I (1993)). Similarly, toxins andvenoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),intracellular receptors (e.g., which mediate the effects of varioussmall ligands, including steroids, thyroid hormone, retinoids andvitamin D; peptides), drugs, lectins, sugars, nucleic acids (both linearand cyclic polymer configurations), oligosaccharides, proteins,phospholipids and antibodies can all interact with various cellreceptors.

Synthetic polymers, such as polyurethanes, polyesters, polycarbonates,polyureas, polyamides, polyethyleneimines, polyarylene sulfides,polysiloxanes, polyimides, and polyacetates can also form an appropriatetag or tag binder. Many other tag/tag binder pairs are also useful inassay systems described herein, as would be apparent to one of skillupon review of this disclosure.

Common linkers such as peptides, polyethers, and the like can also serveas tags, and include polypeptide sequences, such as poly-Gly sequencesof between about 5 and 200 amino acids (SEQ ID NO:42). Such flexiblelinkers are known to those of skill in the art. For example,poly(ethylene glycol) linkers are available from Shearwater Polymers,Inc., Huntsville, Ala. These linkers optionally have amide linkages,sulfhydryl linkages, or heterofunctional linkages.

Tag binders are fixed to solid substrates using any of a variety ofmethods currently available. Solid substrates are commonly derivatizedor functionalized by exposing all or a portion of the substrate to achemical reagent that fixes a chemical group to the surface that isreactive with a portion of the tag binder. For example, groups that aresuitable for attachment to a longer chain portion would include amines,hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes andhydroxyalkylsilanes can be used to functionalize a variety of surfaces,such as glass surfaces. The construction of such solid phase biopolymerarrays is well described in the literature (see, e.g., Merrifield, J.Am. Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of,e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987)(describing synthesis of solid phase components on pins); Frank andDoring, Tetrahedron 44:60316040 (1988) (describing synthesis of variouspeptide sequences on cellulose disks); Fodor et al., Science,251:767-777 (1991); Sheldon et al., Clinical Chemistry 39(4):718-719(1993); and Kozal et al., Nature Medicine 2(7):753759 (1996) (alldescribing arrays of biopolymers fixed to solid substrates).Non-chemical approaches for fixing tag binders to substrates includeother common methods, such as heat, cross-linking by UV radiation, andthe like.

The invention provides in vitro assays for identifying, in a highthroughput format, compounds that can modulate the expression oractivity of a polypeptide of the invention. Control reactions thatmeasure activity of a polypeptide of the invention in a cell in areaction that does not include a potential modulator are optional, asthe assays are highly uniform. Such optional control reactions areappropriate and increase the reliability of the assay. Accordingly, insome embodiments, the methods of the invention include such a controlreaction. For each of the assay formats described, “no modulator”control reactions that do not include a modulator provide a backgroundlevel of binding activity.

In some assays it will be desirable to have positive controls. At leasttwo types of positive controls are appropriate. First, a known activatorof a polypeptide or a polynucleotide of the invention can be incubatedwith one sample of the assay, and the resulting increase in signalresulting from an increased expression level or activity of apolypeptide or a polynucleotide of the invention are determinedaccording to the methods herein. Second, a known inhibitor of apolypeptide or a polynucleotide of the invention can be added, and theresulting decrease in signal for the expression or activity of apolypeptide or a polynucleotide of the invention can be similarlydetected. It will be appreciated that modulators can also be combinedwith activators or inhibitors to find modulators that inhibit theincrease or decrease that is otherwise caused by the presence of theknown modulator of a polypeptide or a polynucleotide of the invention.

VIII. Compositions, Kits and Integrated Systems

The invention provides compositions, kits and integrated systems forpracticing the assays described herein using nucleic acids orpolypeptides of the invention, antibodies, etc.

The invention provides assay compositions for use in solid phase assays;such compositions can include, for example, one or more nucleic acidsencoding a polypeptide of the invention immobilized on a solid support,and a labeling reagent. In each case, the assay compositions can alsoinclude additional reagents that are desirable for hybridization.Modulators of expression or activity of a polypeptide of the inventioncan also be included in the assay compositions.

The invention also provides kits for carrying out the assays of theinvention. The kits typically include a probe that comprises an antibodythat specifically binds to a polypeptide of the invention or apolynucleotide sequence encoding such polypeptides, and a label fordetecting the presence of the probe. The kits may include at least onepolynucleotide sequence encoding a polypeptide of the invention. Kitscan include any of the compositions noted above, and optionally furtherinclude additional components such as instructions to practice ahigh-throughput method of assaying for an effect on expression of thegenes encoding a polypeptide of the invention, or on activity of apolypeptide of the invention, one or more containers or compartments(e.g., to hold the probe, labels, or the like), a control modulator ofthe expression or activity of a polypeptide of the invention, a roboticarmature for mixing kit components or the like.

The invention also provides integrated systems for high-throughputscreening of potential modulators for an effect on the expression oractivity of a polypeptide of the invention. The systems can include arobotic armature which transfers fluid from a source to a destination, acontroller which controls the robotic armature, a label detector, a datastorage unit which records label detection, and an assay component suchas a microtiter dish comprising a well having a reaction mixture or asubstrate comprising a fixed nucleic acid or immobilization moiety.

A number of robotic fluid transfer systems are available, or can easilybe made from existing components. For example, a Zymate XP (ZymarkCorporation; Hopkinton, Mass.) automated robot using a Microlab 2200(Hamilton; Reno, Nev.) pipetting station can be used to transferparallel samples to 96 well microtiter plates to set up several parallelsimultaneous binding assays.

Optical images viewed (and, optionally, recorded) by a camera or otherrecording device (e.g., a photodiode and data storage device) areoptionally further processed in any of the embodiments herein, e.g., bydigitizing the image and storing and analyzing the image on a computer.A variety of commercially available peripheral equipment and software isavailable for digitizing, storing and analyzing a digitized video ordigitized optical image.

One conventional system carries light from the specimen field to acooled charge-coupled device (CCD) camera, in common use in the art. ACCD camera includes an array of picture elements (pixels). The lightfrom the specimen is imaged on the CCD. Particular pixels correspondingto regions of the specimen (e.g., individual hybridization sites on anarray of biological polymers) are sampled to obtain light intensityreadings for each position. Multiple pixels are processed in parallel toincrease speed. The apparatus and methods of the invention are easilyused for viewing any sample, e.g., by fluorescent or dark fieldmicroscopic techniques.

IX. Administration and Pharmaceutical Compositions

Modulators of the polypeptides of the invention (e.g., antagonists oragonists) can be administered directly to the mammalian subject formodulation of activity of a polypeptide of the invention in vivo.Administration is by any of the routes normally used for introducing amodulator compound into ultimate contact with the tissue to be treatedand is well known to those of skill in the art. Although more than oneroute can be used to administer a particular composition, a particularroute can often provide a more immediate and more effective reactionthan another route.

The pharmaceutical compositions of the invention may comprise apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are determined in part by the particular composition beingadministered, as well as by the particular method used to administer thecomposition. Accordingly, there are a wide variety of suitableformulations of pharmaceutical compositions of the present invention(see, e.g., Remington's Pharmaceutical Sciences, 17^(th) ed. 1985)).

The modulators (e.g., agonists or antagonists) of the expression oractivity of the a polypeptide of the invention, alone or in combinationwith other suitable components, can be prepared for injection or for usein a pump device. Pump devices (also known as “insulin pumps”) arecommonly used to administer insulin to patients and therefore can beeasily adapted to include compositions of the present invention.Manufacturers of insulin pumps include Animas, Disetronic and MiniMed.

The modulators (e.g., agonists or antagonists) of the expression oractivity of a polypeptide of the invention, alone or in combination withother suitable components, can be made into aerosol formulations (i.e.,they can be “nebulized”) to be administered via inhalation. Aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for administration include aqueous and non-aqueoussolutions, isotonic sterile solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulationisotonic, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. In the practice of this invention, compositions canbe administered, for example, orally, nasally, topically, intravenously,intraperitoneally, or intrathecally. The formulations of compounds canbe presented in unit-dose or multi-dose sealed containers, such asampoules and vials. Solutions and suspensions can be prepared fromsterile powders, granules, and tablets of the kind previously described.The modulators can also be administered as part of a prepared food ordrug.

The dose administered to a patient, in the context of the presentinvention should be sufficient to induce a beneficial response in thesubject over time. The optimal dose level for any patient will depend ona variety of factors including the efficacy of the specific modulatoremployed, the age, body weight, physical activity, and diet of thepatient, on a possible combination with other drugs, and on the severityof the case of diabetes. It is recommended that the daily dosage of themodulator be determined for each individual patient by those skilled inthe art in a similar way as for known insulin compositions. The size ofthe dose also will be determined by the existence, nature, and extent ofany adverse side-effects that accompany the administration of aparticular compound or vector in a particular subject.

In determining the effective amount of the modulator to be administereda physician may evaluate circulating plasma levels of the modulator,modulator toxicity, and the production of anti-modulator antibodies. Ingeneral, the dose equivalent of a modulator is from about 1 ng/kg to 10mg/kg for a typical subject.

For administration, modulators of the present invention can beadministered at a rate determined by the LD-50 of the modulator, and theside-effects of the modulator at various concentrations, as applied tothe mass and overall health of the subject. Administration can beaccomplished via single or divided doses.

The compounds of the present invention can also be used effectively incombination with one or more additional active agents depending on thedesired target therapy (see, e.g., Turner, N. et al. Prog. Drug Res.(1998) 51: 33-94; Haffner, S. Diabetes Care (1998) 21: 160-178; andDeFronzo, R. et al. (eds.), Diabetes Reviews (1997) Vol. 5 No. 4). Anumber of studies have investigated the benefits of combinationtherapies with oral agents (see, e.g., Mahler, R., J. Clin. Endocrinol.Metab. (1999) 84: 1165-71; United Kingdom Prospective Diabetes StudyGroup: UKPDS 28, Diabetes Care (1998) 21: 87-92; Bardin, C. W., (ed.),Current Therapy In Endocrinology And Metabolism, 6th Edition (Mosby—YearBook, Inc., St. Louis, Mo. 1997); Chiasson, J. et al., Ann. Intern. Med.(1994) 121: 928-935; Coniff, R. et al., Clin. Ther. (1997) 19: 16-26;Coniff, R. et al., Am. J. Med. (1995) 98: 443-451; and Iwamoto, Y. etal., Diabet. Med. (1996) 13 365-370; Kwiterovich, P. Am. J. Cardiol(1998) 82(12A): 3U-17U). These studies indicate that modulation ofdiabetes, among other diseases, can be further improved by the additionof a second agent to the therapeutic regimen. Combination therapyincludes administration of a single pharmaceutical dosage formulationthat contains a modulator of the invention and one or more additionalactive agents, as well as administration of a modulator and each activeagent in its own separate pharmaceutical dosage formulation. Forexample, a modulator and a thiazolidinedione can be administered to thehuman subject together in a single oral dosage composition, such as atablet or capsule, or each agent can be administered in separate oraldosage formulations. Where separate dosage formulations are used, amodulator and one or more additional active agents can be administeredat essentially the same time (i.e., concurrently), or at separatelystaggered times (i.e., sequentially). Combination therapy is understoodto include all these regimens.

One example of combination therapy can be seen in treating pre-diabeticindividuals (e.g., to prevent progression into type 2 diabetes) ordiabetic individuals (or treating diabetes and its related symptoms,complications, and disorders), wherein the modulators can be effectivelyused in combination with, for example, sulfonylureas (such aschlorpropamide, tolbutamide, acetohexamide, tolazamide, glyburide,gliclazide, glynase, glimepiride, and glipizide); biguanides (such asmetformin); a PPAR beta delta agonist; a ligand or agonist of PPAR gammasuch as thiazolidinediones (such as ciglitazone, pioglitazone (see,e.g., U.S. Pat. No. 6,218,409), troglitazone, and rosiglitazone (see,e.g., U.S. Pat. No. 5,859,037)); PPAR alpha agonists such as clofibrate,gemfibrozil, fenofibrate, ciprofibrate, and bezafibrate;dehydroepiandrosterone (also referred to as DHEA or its conjugatedsulphate ester, DHEA-SO4); antiglucocorticoids; TNFα inhibitors;α-glucosidase inhibitors (such as acarbose, miglitol, and voglibose);amylin and amylin derivatives (such as pramlintide, (see, also, U.S.Pat. Nos. 5,902,726; 5,124,314; 5,175,145 and 6,143,718.)); insulinsecretogogues (such as repaglinide, gliquidone, and nateglinide (see,also, U.S. Pat. Nos. 6,251,856; 6,251,865; 6,221,633; 6,174,856)), andinsulin.

XI. Diagnosis of Diabetes

The present invention also provides methods of diagnosing diabetes or apredisposition of at least some of the pathologies of diabetes.Diagnosis can involve determination of a genotype of an individual andcomparison of the genotype with alleles known to have an associationwith the occurrence of diabetes. Alternatively, diagnosis also involvesdetermining the level of a polypeptide or polynucleotide of theinvention in a patient and then comparing the level to a baseline orrange. Typically, the baseline value is representative of a polypeptideor polynucleotide of the invention in a healthy (e.g., non-diabeticlean) person.

Variation of levels (e.g., low or high levels) of a polypeptide orpolynucleotide of the invention compared to the baseline range indicatesthat the patient is either diabetic or at risk of developing at leastsome of the pathologies of diabetes (e.g., pre-diabetic). The level of apolypeptide in a non-diabetic lean individual can be a reading from asingle individual, but is typically a statistically relevant averagefrom a group of lean individuals. The level of a polypeptide in a leanindividual can be represented by a value, for example in a computerprogram.

In some embodiments, the baseline level and the level in a lean samplefrom an individual, or at least two samples from the same individualdiffer by at least about 5%, 10%, 20%, 50%, 75%, 100%, 150%, 200%, 300%,400%, 500%, 1000% or more. In some embodiments, the sample from theindividual is greater by at least one of the above-listed percentagesrelative to the baseline level. In some embodiments, the sample from theindividual is lower by at least one of the above-listed percentagesrelative to the baseline level.

Glucose/insulin tolerance tests can also be used to detect the effect ofglucose levels on levels of a polypeptide or polynucleotide of theinvention. In glucose tolerance tests, the patient's ability to toleratea standard oral glucose load is evaluated by assessing serum and urinespecimens for glucose levels. Blood samples are taken before the glucoseis ingested, glucose is given by mouth, and blood or urine glucoselevels are tested at set intervals after glucose ingestion. Similarly,meal tolerance tests can also be used to detect the effect of insulin orfood, respectively, on levels of a polypeptide or polynucleotide of theinvention.

EXAMPLE

The following example is offered to illustrate, but not to limit theclaimed invention.

IC-RFX is a previously undescribed member of the RFX family that sharesthe conserved DNA binding domain (DBD) with the rest of the members ofthe RFX family (FIG. 4). IC-RFX also has conserved (with RFXs 1-4)dimerization, B and C domains. See, Emery P et al., Nucleic Acids Res.24: 803-807 (1996) and FIGS. 5-8. There is no obvious A domain inIC-RFX. In this regard, IC-RFX most resembles RFX4 in domainorganization (Morotomi-Yano K et al., J. Biol. Chem. 277: 836-842(2002)). Indeed, the RFX4 DBD also has the greatest amino acid identitywith that of IC-RFX (54/76 or 71%) (FIG. 5).

IC-RFX is also similar to the C. elegans RFX protein daf-19 in B, C,dimerization and DBD domains (45/76 or 59% identical in the DBD domain)and in overall domain organization. Daf-19 is specifically expressed ina subset of sensory neurons, and these neurons are absent in daf-19 nullmutants. See, e.g., Swoboda P et al. Mol. Cell. 5: 411-2 (2000)). Daf-19is the only RFX protein found in C. elegans. RFX1, 2, 3, 4, and 5 areall transcription factors that bind to conserved DNA sequences called Xboxes in the promoters of the genes that they regulate (Sengupta K etal., J. Biol. Chem. 277: 24926-24937 (2002)).

Analysis of rat, mouse and human islet EST databases and expressionprofiling on custom islet Affymetrix oligonucleotide arrays revealedthat ESTs and Affymetrix probe sets corresponding to IC-RFX are highlyenriched in pancreatic islets and beta cells and are not detected in anyother tissue examined (FIG. 1 and FIG. 3). These negative tissuesinclude whole pancreas (islets compose less than 5% of total pancreasmRNA), adipose, brain, heart, kidney, liver, lung, skeletal muscle,small intestine or thymus, pituitary). Northern blots of human RNAsconfirmed the specific expression of IC-RFX in islets (FIG. 2).

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. An antisense polynucleotide or a small interfering RNA (siRNA) thatinhibits expression of a nucleic acid encoding a polypeptide comprisingin the following order: a proline/glutamine rich domain, an RFX DNAbinding domain (SEQ ID NO:4), an RFX B domain (SEQ ID NO:5), an RFX Cdomain (SEQ ID NO:6), a dimerization domain (SEQ ID NO:7) and aserine/threonine domain.
 2. The antisense polynucleotide or siRNA ofclaim 1, wherein the nucleic acid encodes SEQ ID NO:2.
 3. The antisensepolynucleotide or siRNA of claim 1, wherein the nucleic acid comprisesSEQ ID NO:1.
 4. The antisense polynucleotide or siRNA of claim 1, whichis an antisense polynucleotide.
 5. The antisense polynucleotide or siRNAof claim 1, which is an siRNA.
 6. An expression vector encoding theantisense polynucleotide or siRNA of claim
 1. 7. The expression vectorof claim 6, which encodes the antisense polynucleotide.
 8. Theexpression vector of claim 6, which encodes the siRNA.
 9. The expressionvector of claim 6, wherein the nucleic acid encodes SEQ ID NO:2.
 10. Theexpression vector of claim 6, wherein the nucleic acid comprises SEQ IDNO:1.
 11. A host cell transfected with the expression vector of claim 6.12. The host cell of claim 11, wherein the cell is a pancreatic isletcell.
 13. The host cell of claim 12, wherein the cell is an isletβ-cell.